Spiro-oxindole mdm2 antagonists

ABSTRACT

Provided herein are compounds, compositions, and methods in the field of medicinal chemistry. The compounds and compositions provided herein relate to spiro-oxindoles which function as antagonists of the interaction between p53 and MDM2, and their use as therapeutics for the treatment of cancer and other diseases.

BACKGROUND

The aggressive cancer cell phenotype is the result of a variety ofgenetic and epigenetic alterations leading to deregulation ofintracellular signaling pathways (Ponder, Nature 411:336 (2001)). Cancercells typically fail to execute an apoptotic program, and lack ofappropriate apoptosis due to defects in the normal apoptosis machineryis considered a hallmark of cancer (Lowe et al., Carcinogenesis 21:485(2000)). The inability of cancer cells to execute an apoptotic programdue to defects in the normal apoptotic machinery is often associatedwith an increase in resistance to chemotherapy, radiation, orimmunotherapy-induced apoptosis. Primary or acquired resistance of humancancer of different origins to current treatment protocols due toapoptosis defects is a major problem in current cancer therapy (Lowe etal., Carcinogenesis 21:485 (2000); Nicholson, Nature 407:810 (2000)).Accordingly, current and future efforts towards designing and developingnew molecular target-specific anticancer therapies to improve survivaland quality of life of cancer patients must include strategies thatspecifically target cancer cell resistance to apoptosis.

The p53 tumor suppressor plays a central role in controlling cell cycleprogression, senescence, and apoptosis (Vogelstein et al., Nature408:307 (2000); Goberdhan, Cancer Cell 7:505 (2005)). MDM2 and p53 arepart of an auto-regulatory feed-back loop (Wu et al., Genes Dev. 7:1126(1993)). MDM2 is transcriptionally activated by p53 and MDM2, in turn,inhibits p53 activity by at least three mechanisms (Wu et al., GenesDev. 7:1126 (1993). First, MDM2 protein directly binds to the p53transactivation domain and thereby inhibits p53-mediatedtransactivation. Second, MDM2 protein contains a nuclear export signalsequence, and upon binding to p53, induces the nuclear export of p53,preventing p53 from binding to the targeted DNAs. Third, MDM2 protein isan E3 ubiquitin ligase and upon binding to p53 is able to promote p53degradation.

Although high-affinity peptide-based inhibitors of MDM2 have beensuccessfully designed in the past (Garcia-Echeverria et al., Med. Chem.43:3205 (2000)), these inhibitors are not suitable therapeutic moleculesbecause of their poor cell permeability and in vivo bioavailability.Despite intensive efforts by the pharmaceutical industry, highthroughput screening strategies have had very limited success inidentifying potent, non-peptide small molecule inhibitors. Accordingly,there is a need for non-peptide, drug-like, small molecule inhibitors ofthe p53-MDM2 interaction.

The structural basis of the interaction p53 and MDM2 has beenestablished by x-ray crystallography (Kussie et al., Science 274:948(1996)).

Spiro-oxindole-based antagonists of the p53-MDM2 interaction aredescribed in U.S. Pat. Nos. 7,759,383 B2 and 7,737,174 B2.

Skin cancer or melanoma is a commonly found type of cancer. Even thoughmelanoma represents only a small fraction of the total number of cancercases, it is responsible for many cancer deaths. According to statisticsprovided by the American Cancer Society, in contrast to many other typesof cancers, the number of new cases of melanoma in the United States isstill on the rise.

As with all cancers, it is imperative to diagnose melanoma early. About70% of melanomas are “superficial spreading”, meaning that they undergoa superficial, radial growth phase before they grow vertically andinvade underlying tissue, a much more serious condition. Unfortunately,about 20% of cutaneous melanomas immediately start out with a verticalgrowth phase, which explains why these tumors are so dangerous. The5-year survival rate for Stage 1 melanoma is very good. However, thisdrops off rapidly when cancer is allowed to progress and invade, firstlocally and then more distantly. Survival rate for Stage 2 disease isonly 40-80%, Stage 3 10-70% and Stage 4 is almost invariably lethalwithin 5 years (<5-10% survives beyond 5 years) due to untreatabledistant metastasis to especially lung and brain.

Melanoma originates from malignant transformation of melanocytes, thepigment producing skin cells, via atypical and dysplastic premalignantintermediate stages to locally invasive and finally metastatic melanoma.A large number of genes have been implicated to play a role in theseprocesses. Metastatic melanoma, the usual cause of death, is notoriouslyresistant to conventional therapy.

SUMMARY OF THE INVENTION

The present disclosure contemplates that exposure of humans and animalsto therapeutically effective amounts of drug(s) (e.g., small molecules)that increase the function(s) of p53 and p53-related proteins (e.g.,p63, p73) inhibits the growth of p53 expressing cells. In someembodiments, the compounds provided herein inhibit the interactionbetween p53 or p53-related proteins and MDM2 or MDM2-related proteins(e.g., MDMX). Inhibiting the interaction between p53 or p53-relatedproteins and MDM2 or MDM2-related proteins inhibits the growth of cells.For example, inhibiting the interaction between p53 or p53-relatedproteins and MDM2 or MDM2-related proteins can inhibit cancer cells orsupporting cells and/or renders such cells as a population moresusceptible to the cell death-inducing activity of cancer therapeuticdrugs or radiation therapies. In some embodiments, the inhibitorsprovided herein prolong the half-life of p53 by interfering with thep53-MDM2 interaction that would normally promote degradation of p53. Thecompounds provided herein satisfy an unmet need for the treatment ofmultiple cancer types, either when administered as monotherapy to inducesenescence, cell growth inhibition, apoptosis and/or cell cycle arrestin cancer cells, or when administered in a temporal relationship withadditional agent(s), such as other cell death-inducing or cell cycledisrupting cancer therapeutic drugs or radiation therapies (combinationtherapies), so as to render a greater proportion of the cancer cells orsupportive cells susceptible to executing the apoptosis program comparedto the corresponding proportion of cells in an animal treated only withthe cancer therapeutic drug or radiation therapy alone.

In some embodiments, treatment of animals (including humans) with atherapeutically effective amount of one or more compounds providedherein and an anticancer agent produces a greater anti-tumor activityand clinical benefit in such animals compared to those treated with thecompound or anticancer drugs/radiation alone. Put another way, becausethe compounds provided herein can lower the apoptotic threshold of cellsthat express p53 or p53-related protein, the proportion of cells thatsuccessfully execute the apoptosis program in response to the apoptosisinducing activity of anticancer drugs/radiation will be increased whenused in combination with one or more of the compounds provided herein.Alternatively, the compounds provided herein can be used to allowadministration of a lower, and therefore less toxic and more tolerable,dose of an anticancer drug and/or radiation to produce the same tumorresponse/clinical benefit as the conventional dose of the anticancerdrug/radiation alone. Since the doses for all approved anticancer drugsand radiation treatments are known, the present compounds, compositions,and methods provided herein can be used with one or more approvedanticancer drugs and/or radiation treatment. Also, since the compoundsprovided herein may act at least in part by stimulating thepro-apoptotic and/or cell cycle-inhibiting activities of p53 andp53-related proteins, the exposure of cancer cells and supporting cellsto therapeutically effective amounts of the compounds can be temporallylinked to coincide with the attempts of cells to execute the apoptosisprogram in response to the anticancer drug or radiation therapy. Thus,in some embodiments, administering the compounds or compositionsprovided herein in combination with other known anticancer drugs provideespecially efficacious therapeutic practices.

In other embodiments, the inhibitors of the interaction between p53 orp53-related proteins and MDM2 and MDM2-related proteins provided hereinmay protect normal (e.g., non-hyperproliferative) cells from the toxiceffects of certain chemotherapeutic agents and radiation, possiblythrough the ability of the inhibitors to induce cell cycle arrest ofnormal cells. For example, the inhibitors provided herein may cause cellcycle arrest in cells comprising wild-type or functional p53 (and/orwild-type or functional p53-related proteins) while having no or lesseffect on cancer cells comprising mutated, deleted, or otherwise non- orless functional p53 (and/or mutated, deleted, or otherwise non- or lessfunctional p53-related proteins). This differential protective effectmay allow for more effective treatment of cancer by allowing the use ofhigher doses or longer treatments of chemotherapeutic agents ortreatments without increasing the toxic side effects of such treatmentwhen administered in combination with inhibitors provided herein.

Applicants have found that certain spiro-oxindoles provided hereindisplay an unexpected combination of drug-like properties. Theunexpected combinations include, e.g., two or more of in vitro efficacy,in vivo efficacy, in vitro liver microsome stability, desirableabsorption, distribution, metabolism, and excretion (ADME) properties.For example, certain spiro-oxindoles provided herein are more resistantto metabolic degradation e.g., as measured by in vitro liver microsomalstability and/or in vivo pharmacokinetics, and/or display improved invivo efficacy as compared to known antagonists of the p53-MDM2interaction.

Applicants have also found that metabolically cleavable groups can beused to increase the aqueous solubility of the parent molecule. Thus, insome embodiments, the spiro-oxindoles provided herein are usefulprodrugs with improved aqueous solubility relative to the parentmolecule.

In some embodiments, the compounds provided herein are spiro-oxindoleshaving Formulae I-XXXV (see below under “Compounds”), orpharmaceutically acceptable salts, solvates, or prodrugs thereof. Insome embodiments, the compounds provided herein inhibit the interactionbetween p53 or p53-related proteins and MDM2 or MDM2-related proteins.

In some embodiments, the compounds provided herein contain ametabolically cleavable group. In particular, in some embodiments, thecompounds provided herein contain a hydroxy group of a hydroxycycloalkylside chain that can be used to attach a metabolically cleavable group.Suitable metabolically cleavable groups include, but are not limited to,amino acid esters or phosphate esters.

In some embodiments, the compounds provided herein can be used to inducesenescence, cell cycle arrest and/or apoptosis in cells containingfunctional p53 or p53-related proteins. Also provided herein are methodsof using the compounds provided herein for sensitizing cells toadditional agent(s), such as inducers of senescence, apoptosis and/orcell cycle arrest. The compounds provided herein can also be used toprovide chemoprotection of normal cells through the induction of cellcycle arrest prior to treatment with chemotherapeutic agents. In oneembodiment, the methods of rendering a normal cell resistant tochemotherapeutic agents or treatments comprises contacting the cell withone or more compounds provided herein. In one embodiment, methods ofprotecting normal cells in an animal having a hyperproliferative diseasefrom the toxic side effects of chemotherapeutic agents or treatments,comprises administering to the animal a compound provided herein.Provided herein are methods for the treatment, amelioration, orprevention of disorders, side effects, or conditions caused by theadministration of chemotherapeutic agents to normal cells comprisingadministering to an animal undergoing chemotherapy a compound providedherein. Examples of such disorders and conditions caused by chemotherapyinclude, without limitation, mucositis, stomatitis, xerostomia,gastrointestinal disorders, and alopecia.

The compounds provided herein are useful for the treatment,amelioration, or prevention of disorders, such as those responsive toinduction of apoptotic cell death, e.g., disorders characterized bydysregulation of apoptosis, including hyperproliferative diseases suchas cancer. In certain embodiments, the compounds can be used to treat,ameliorate, or prevent cancer that is characterized by resistance tocancer therapies (e.g., those cancer cells which are chemoresistant,radiation resistant, hormone resistant, and the like). In otherembodiments, the compounds can be used to treat hyperproliferativediseases characterized by expression of functional p53 or p53-relatedproteins. In other embodiments, the compounds provided herein can beused to protect normal (e.g., non-hyperproliferative) cells from thetoxic side effects of chemotherapeutic agents and treatments by theinduction of cell cycle arrest in those cells.

In one embodiment, pharmaceutical compositions are provided. Thepharmaceutical compositions can comprise one of more of the compoundsprovided herein and a pharmaceutically acceptable carrier.

In one embodiment, kits are provided. The kits can comprise one or moreof the compounds provided herein, or a pharmaceutically acceptable saltthereof, and instructions for administering the compound to an animal.The kits may optionally contain other therapeutic agents, e.g.,anticancer agents or apoptosis-modulating agents.

In one embodiment, methods of treating, preventing, or ameliorating ahyperproliferative disease, e.g., cancer, in a patient comprisingpulsatile administration to the patient a therapeutically effectiveamount of one or more of the compounds provided herein, orpharmaceutically acceptable salts, solvates, or prodrugs thereof areprovided.

In one embodiment, methods of treating, preventing, or ameliorating ahyperproliferative disease, e.g., cancer, in a patient comprisingpulsatile administration to the patient a therapeutically effectiveamount of one or more of the compounds provided herein, orpharmaceutically acceptable salts, solvates, or prodrugs thereof, incombination with one or more additional therapeutic, e.g., anticancer,agents.

In one embodiment, kits comprising one or more of the compounds providedherein, or pharmaceutically acceptable salts, solvates, or prodrugsthereof, and instructions for administering the compound(s) to a patienthaving a hyperproliferative disease by pulsatile dosing are provided.The kits can optionally contain one or more additional therapeutic,e.g., anticancer, agents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a reverse phase HPLC chromatogram of MI-519-64 after isolationby column chromatography on silica gel.

FIG. 2 is reverse phase HPLC chromatogram of MI-519-64 after treatmentwith acetonitrile/water for 12 h. Three isomers are present. MI-519-64and MI-519-6401 correspond to RP-HPLC peaks at 30.578 minutes, and31.787 minutes, respectively. The isomer eluting at 29.162 minutes isreferred to as MI-519-6402.

FIG. 3 is reverse phase HPLC chromatogram of MI-519-64 after treatmentwith acetonitrile/water for 3 days.

FIG. 4 is a line graph showing the binding affinities of MI-519-64,MI-519-6401, and MI-519-6402 to human MDM2 protein, as determined usinga fluorescence-polarization binding assay. The purity of each isomerused in this experiment (as determined by RP-HPLC) are as follows:MI-519-6402: 90% (with 10% of MI-519-64); MI-519-64: 93% (with 3% ofMI-519-64 and 4% of MI-519-6401); and MI-519-6401: >99%. The log IC₅₀values for MI-519-6402, MI-519-64, and MI-519-6401 are 2.030 nM, 1.598nM, and 0.8354 nM, respectively.

FIG. 5 is a line graph showing the binding affinities of MI-773 (TFAsalt), MI-77301 (TFA salt), MI-77301 (free amine), and MI-77302 (TFAsalt) to human MDM2 protein, as determined using afluorescence-polarization binding assay.

FIG. 6 is a line graph showing the stability of MI-773 (TFA salt) atvarious time points in water/methanol=1:1 with 0.1% of TFA, pH 2.1. Thecompound corresponding to peak 1 is MI-77302. The compound correspondingto peak 3 is MI-773. The compound corresponding to peak 4 is MI-77301.

FIG. 7 is a line graph showing the stability of MI-77301 (TFA salt) atvarious time points in water/methanol=1:1 with 0.1% of TFA, pH 2.1. Thecompound corresponding to peak 1 is MI-77302. The compound correspondingto peak 3 is MI-773. The compound corresponding to peak 4 is MI-77301.

FIG. 8 is an illustration showing western blot analysis of p53activation and apoptosis induced by MI-773 and MI-77301 in the SJSA-1(osteosarcoma) cell line.

FIG. 9 is an illustration showing western blot analysis of p53activation and apoptosis induced by MI-519-64 and MI-519-6401 in theSJSA-1 cell line.

FIG. 10 is a bar graph showing apoptosis induced by MI-773 and MI-77301in the SJSA-1 cell line.

FIG. 11 is a bar graph showing cell death induced by MI-519-64 andMI-519-6401 in the SJSA-1 cell line.

FIG. 12 is a bar graph showing cell death induced by MI-519-64 andMI-519-6401 in the RS4;11 (human acute lymphoblastic leukemia (ALL))cell line.

FIG. 13 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401in SJSA-1 tumors in mice.

FIG. 14 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401in RS4;11 tumors in mice.

FIG. 15 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-773 and MI-77301 inSJSA-1 tumors in mice.

FIG. 16 is an illustration showing three western blot analyses of p53activation and apoptosis induced by MI-773 and MI-77301 in the RS4;11cell line.

FIG. 17 is a line graph showing in vivo antitumor activity of MI-519-64,MI-519-6401, MI-773, and MI-77301 in the SJSA-1 osteosarcoma xenograftmodel in mice.

FIG. 18 is a line graph showing the animal weight followingadministration of MI-519-64, MI-519-6401, MI-773, and MI-77301 in mice.

FIG. 19 is a line graph showing in vivo antitumor activity ofMI-519-6401 and MI-77301 in the 22Rv1 human prostate xenograft model inmice.

FIG. 20 is a line graph showing in vivo antitumor activity of MI-77301in the SJSA-1 osteosarcoma xenograft model in mice (Cpd-B=MI-77301;treatment schedule=QD11).

FIG. 21 is a line graph showing the animal weight followingadministration of MI-77301 in mice (Cpd-B=MI-77301).

FIG. 22 is a line graph showing in vivo antitumor activity in the SJSA-1osteosarcoma xenograft model in mice (Cpd-B=MI-77301).

FIG. 23 is a line graph showing in vivo antitumor activity of MI-77301in the SJSA-1 osteosarcoma xenograft model in mice (Cpd-B=MI-77301).

FIG. 24 is a line graph showing the cell growth inhibition activity ofMI-77301 in melanoma cell lines.

FIG. 25 is an illustration showing western blot analysis of p53activation induced by MI-77301 in SK-Mel-103 (human melanoma) cells.

FIG. 26 is an illustration showing western blot analysis of p53activation induced by MI-77301 in UACC-62 (p53 wt melanoma) andSK-Mel-19 (human melanoma) cells.

FIG. 27 is a bar graph showing apoptosis induced by MI-77301 in theUACC-62 cell line.

FIG. 28 is an illustration showing western blot analysis of in vivoactivation of p53 induced by MI-773001 in SK-Mel-103 melanoma xenograftsin mice.

FIG. 29 is a line graph showing in vivo antitumor activity of MI-77301in the SK-Mel-103 melanoma xenograft model in mice.

FIG. 30 is reverse phase HPLC chromatogram of substantially pureCB061—Isomer B.

FIG. 31 is a line graph showing in vivo antitumor activity of MI-77301in the HCT-116 human colorectal tumor xenograft model in mice.

FIG. 32 is a line graph showing in vivo antitumor activity of MI-77301in the LNCAP human prostate tumor xenograft model in mice.

FIG. 33 is a line graph showing in vivo antitumor activity of MI-77301in the RS4;11 human acute lymphoblastic leukemia xenograft model inmice.

FIG. 34 is a series of three ¹³C CPMAS NMR spectrograms showing MI-77301(top), MI-773 (middle), and MI-77302 (bottom).

FIG. 35 is a reverse phase HPLC chromatogram of substantially pureMI-773 (eluent: MeOH/water with 0.1% TFA).

FIG. 36 is a reverse phase HPLC chromatogram of substantially pureMI-77301 (eluent: MeOH/water with 0.1% TFA).

FIG. 37 is a line graph showing the stability of MI-773 (TFA salt) atvarious time points in water/methanol=1:1 with 0.1% of TEA, pH 10.8. Thecompound corresponding to peak 3 is MI-773. The compound correspondingto peak 4 is MI 77301.

FIG. 38 is a line graph showing the stability of MI-773 (TFA salt) atvarious time points in water/methanol=1:1, pH 3.9. The compoundcorresponding to peak 3 is MI-773. The compound corresponding to peak 4is MI-77301.

FIG. 39 is reverse phase HPLC chromatogram of substantially pure C027(eluent: acetonitrile/H₂O with 0.1% TFA).

FIG. 40 is reverse phase HPLC chromatogram of substantially pure C02701(eluent: acetonitrile/H₂O with 0.1% TFA).

FIG. 41 is reverse phase HPLC chromatogram of C029 substantially free ofother stereoisomers (eluent: acetonitrile/H₂O with 0.1% TFA).

FIG. 42 is reverse phase HPLC chromatogram of substantially pure C02901(eluent: acetonitrile/H₂O with 0.1% TFA).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that inhibit the interaction between p53or p53-related proteins and MDM2 or MDM2-related proteins. By inhibitingthe negative effect of MDM2 or MDM2-related proteins on p53 orp53-related proteins, these compounds sensitize cells to inducers ofapoptosis and/or cell cycle arrest. In some embodiments, the compoundsprovided herein induce apoptosis and/or cell cycle arrest. Therefore,also provided herein are methods of sensitizing cells to inducers ofapoptosis and/or cell cycle arrest and to methods of inducing apoptosisand/or cell cycle arrest in cells. In some embodiments, the methodscomprise contacting the cells with one or more compounds provided hereinalone or in combination with additional agent(s), e.g., an inducer ofapoptosis or a cell cycle disrupter.

Also provided herein are methods of treating, ameliorating, orpreventing disorders, e.g., a hyperproliferative disease, e.g., cancer,in an patient, comprising administering to the patient one or morecompounds provided herein and additional agent(s), e.g., an inducer ofapoptosis. Such disorders include those characterized by a dysregulationof apoptosis and those characterized by the proliferation of cellsexpressing functional p53 or p53-related proteins. In other embodiments,methods of protecting normal (e.g., non-hyperproliferative) cells in ananimal from the toxic side effects of chemotherapeutic agents andtreatments are provided. The methods comprise administering to theanimal one or more compounds provided herein. Also provided herein aremethods of treating preventing, or ameliorating a hyperproliferativedisease, e.g., cancer, in a patient comprising administering to thepatient a therapeutically effective amount of one or more of thecompounds provided herein, or pharmaceutically acceptable salts,solvates, or prodrugs thereof, according to a pulsatile dosing regimen.

Also provided herein are methods of treating preventing, or amelioratinga hyperproliferative disease, e.g., cancer, in a patient comprisingadministering to the patient a therapeutically effective amount of oneor more of the compounds provided herein, or pharmaceutically acceptablesalts, solvates, or prodrugs thereof, according to a pulsatile dosingregimen in combination with one or more additional therapeutic, e.g.,anticancer, agents.

Also provided herein are kits comprising one or more of the compoundsprovided herein, and instructions for administering the compound(s) to apatient having a hyperproliferative disease by pulsatile dosing. Thekits can optionally contain one or more additional therapeutic, e.g.,anticancer, agents

DEFINITIONS

The terms “pulsatile administration,” “pulsatile dose administration,”or “pulsatile dosing” as used herein, refer to intermittent (i.e., notcontinuous) administration of one or more of the compounds providedherein, or pharmaceutically acceptable salts, solvates, or prodrugsthereof, to a patient. Pulsatile dose administration regimens useful inthe present disclosure encompass any discontinuous administrationregimen that provides a therapeutically effective amount of thecompound(s) provided herein, or pharmaceutically acceptable salts,solvates, or prodrugs thereof, to a patient in need thereof. Pulsatiledosing regimens can use equivalent, lower, or higher doses of compounds,or pharmaceutically acceptable salts, solvates, or prodrugs thereof,than would be used in continuous dosing regimens. The compounds providedherein, or pharmaceutically acceptable salts, solvates, or prodrugsthereof, can be administered as a single agent under a pulsatile dosingregimen or can be administered under a pulsatile dosing regimen incombination with one or more additional anticancer agents (where theadditional anticancer agents are administered either on a continuous ora pulsatile regimen). On the day that compounds, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are scheduled to beadministered to the patient, administration can occur in a single or individed doses, e.g., once-a-day, twice-a-day, three times a day, fourtimes a day or more. In one embodiment, compounds provided herein, orpharmaceutically acceptable salts, solvates, or prodrugs thereof, areadministered once (QD) or twice (BID) on the day it is schedule to beadministered. In one embodiment the compounds provided, orpharmaceutically acceptable salts, solvates, or prodrugs thereof, areadministered orally to the patient according to a pulsatile dosingregimen. In one embodiment the compounds provided herein, orpharmaceutically acceptable salts, solvates, or prodrugs thereof, areadministered intravenously to the patient according to a pulsatiledosing regimen.

The therapeutic utility of drug administration can be offset by thenumber and severity of adverse events a patient experiences. Pulsatiledosing of compounds provided herein, or pharmaceutically acceptablesalts, solvates, or prodrugs thereof, can result in a reduction in thenumber and/or severity of clinical adverse events coupled with amaintenance or enhancement in clinical efficacy, as compared tocontinuous daily dosing. The clinical benefits of pulsatile doseadministration of compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, can be more prominentwhen combined with the administration of other therapeutic agents to thepatient.

In one embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered to apatient no more frequently than one day out of every two days (e.g.,administration occurs on day 1, day 3, day 5, day 7, day 9, etc.), oneout of every three days (e.g., administration occurs on day 1, day 4,day 7, day 10, etc.), one out of every four days, one out of every fivedays, one out of every six days, one out of every seven days, one out ofevery eight days, one out of every nine days, one out of every ten days,one out of every two weeks, one out of every three weeks, one out ofevery four weeks, one out of every five weeks, or longer. The pulsatiledosing regimen can continue for one, two, three or four weeks, one, two,three or four months, one, two, three or four years or longer.

In another embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered to apatient one day a week, e.g., a compound of Formulae I-XXXV, or apharmaceutically acceptable salt, solvate, or prodrug thereof, isadministered to a patient on one day followed by six consecutive dayswherein the compound is not administered. In another embodiment,compounds having Formulae I-XXXV, or pharmaceutically acceptable salts,solvates, or prodrugs thereof, administered to a patient one day everytwo weeks. In another embodiment, compounds having Formulae I-XXXV, orpharmaceutically acceptable salts, solvates, or prodrugs thereof, areadministered to a patient one day every three weeks. In anotherembodiment, compounds having Formulae I-XXXV, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered to apatient one day every four weeks.

In another embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered to apatient on a least two consecutive days, e.g., at least three, four,five, six or seven consecutive days, followed by at least one day, atleast two consecutive days, at least three consecutive days, at leastfour consecutive days, at least five consecutive days, at least sixconsecutive days, at least seven consecutive days, at least eightconsecutive days, at least nine consecutive days, at least tenconsecutive days, at least eleven consecutive days, at least twelveconsecutive days, at least thirteen consecutive days, at least twoconsecutive weeks, at least three consecutive weeks, or at least fourconsecutive weeks or longer wherein the compound disclosed herein is notadministered.

In one embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, and one or moreanticancer agents are administered to a patient on day 1 of ananticancer treatment cycle. Typically, the length of the treatment cycleis determined in accord with the approved dosing protocol(s) of the oneor more anticancer agents that are to be administered to the patient incombination with the compounds having Formulae I-XXXV, orpharmaceutically acceptable salts, solvates, or prodrugs thereof. In oneembodiment, the treatment cycle is about 14 days, about 21 days, orabout 28 days. In a particular embodiment, the treatment cycle is 21days. In one embodiment, the treatment cycle is repeated one or moretimes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, or more times.

In another embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered to thepatient on day 1, on days 1 and 2, or on days 1, 2, and 3 of a treatmentcycle and one or more anticancer agents are administered starting on day1 of the treatment cycle in accord with the recommended dosing scheduleof the anticancer agent. In one embodiment, the anticancer agent is achemotherapeutic agent. In another embodiment, the anticancer agent isradiation therapy.

In another embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administered viathe sequential use of a combination of two or more pulsatile dosingschedules. The combination may comprise the same pulsatile dosingschedules or different pulsatile dosing schedules. The sequential use ofa combination of two or more pulsatile dosing regimens may be repeatedas many times as necessary to achieve or maintain a therapeuticresponse, e.g., from one to about fifty times, e.g., from one to abouttwenty times, e.g., from about one to about ten times. With everyrepetition any additional therapeutic agents may be the same ordifferent from that used in the previous repetition.

In another embodiment, compounds provided herein, or pharmaceuticallyacceptable salts, solvates, or prodrugs thereof, are administeredaccording to a pulsatile dosing schedule and/or sequential combinationof two or more pulsatile dosing schedules followed by a waiting period.The term “waiting period,” as used herein, refers to a period of timebetween dosing schedules when a compound disclosed herein is notadministered to the patient. The waiting period may be one, two, three,four, five or six days, one, two or three weeks, one, two, three or fourmonths, one, two, three or four years or longer. In certain embodiments,the waiting period may be one to thirty days, e.g., seven, fourteen,twenty one or thirty days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30days. After the waiting period, the same or a different pulsatile dosingschedule and/or sequential combination of one or more pulsatile dosingschedules of a compound disclosed herein can resume. The pulsatiledosing/waiting period regimen may be repeated as many times as necessaryto achieve or maintain a therapeutic response, e.g., from one to aboutfifty times, e.g., from one to about twenty times, e.g., from about oneto about ten times. With every repetition any additional therapeuticagents may be the same or different from that used in the previousrepetition.

The term “anticancer agent” as used herein, refers to any therapeuticagent (e.g., chemotherapeutic compound and/or molecular therapeuticcompound), antisense therapy, radiation therapy, or surgicalintervention, used in the treatment of hyperproliferative diseases suchas cancer (e.g., in mammals, e.g., in humans).

The term “prodrug” as used herein, refers to a pharmacologicallyinactive derivative of a parent “drug” molecule that requiresbiotransformation (e.g., either spontaneous or enzymatic) within thetarget physiological system to release, or to convert (e.g.,enzymatically, physiologically, mechanically, electromagnetically) theprodrug into the active drug. Prodrugs are designed to overcome problemsassociated with stability, water solubility, toxicity, lack ofspecificity, or limited bioavailability. Exemplary prodrugs comprise anactive drug molecule itself and a chemical masking group (e.g., a groupthat reversibly suppresses the activity of the drug). Some prodrugs arevariations or derivatives of compounds that have groups cleavable undermetabolic conditions. Prodrugs can be readily prepared from the parentcompounds using methods known in the art, such as those described in ATextbook of Drug Design and Development, Krogsgaard-Larsen and H.Bundgaard (eds.), Gordon & Breach, 1991, particularly Chapter 5: “Designand Applications of Prodrugs”; Design of Prodrugs, H. Bundgaard (ed.),Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan(ed.), Marcel Dekker, 1998; Methods in Enzymology, K. Widder et al.(eds.), Vol. 42, Academic Press, 1985, particularly pp. 309-396;Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff(ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. 172-178 andpp. 949-982; Pro-Drugs as Novel Delivery Systems, T. Higuchi and V.Stella (eds.), Am. Chem. Soc., 1975; and Bioreversible Carriers in DrugDesign, E. B. Roche (ed.), Elsevier, 1987.

Exemplary prodrugs become pharmaceutically active in vivo or in vitrowhen they undergo solvolysis under physiological conditions or undergoenzymatic degradation or other biochemical transformation (e.g.,phosphorylation, hydrogenation, dehydrogenation, glycosylation).Prodrugs often offer advantages of water solubility, tissuecompatibility, or delayed release in the mammalian organism. (See e.g.,Bundgaard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam(1985); and Silverman, The Organic Chemistry of Drug Design and DrugAction, pp. 352-401, Academic Press, San Diego, Calif. (1992)). Commonprodrugs include acid derivatives such as esters prepared by reaction ofparent acids with a suitable alcohol (e.g., a lower alkanol) or estersprepared by reaction of parent alcohol with a suitable carboxylic acid,(e.g., an amino acid), amides prepared by reaction of the parent acidcompound with an amine, basic groups reacted to form an acylated basederivative (e.g., a lower alkylamide), or phosphorus-containingderivatives, e.g., phosphate, phosphonate, and phosphoramidate esters,including cyclic phosphate, phosphonate, and phosphoramidate, see, e.g.,US 2007/0249564 A1.

The term “metabolically cleavable group” as used herein, refers togroups which can be cleaved from the parent molecule by metabolicprocesses and be substituted with hydrogen. Certain compounds containingmetabolically cleavable groups may be prodrugs, i.e., they arepharmacologically inactive. Certain other compounds containingmetabolically cleavable groups may be antagonists of the interactionbetween p53 and MDM2. In such cases, these compounds may have more,less, or equivalent activity of the parent molecule. Examples ofmetabolically cleavable groups include those derived from amino acids(see, e.g., US 2006/0241017 A1; US 2006/0287244 A1; and WO 2005/046575A2) or phosphorus-containing compounds (see, e.g., U.S. 2007/0249564 A1)as illustrated in Scheme 1.

The term “pharmaceutically acceptable salt” as used herein, refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound provided herein that is physiologically tolerated in the targetanimal (e.g., a mammal) or human. Salts of the compounds of providedherein may be derived from inorganic or organic acids and bases.Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds provided herein andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds provided hereincompounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄ ⁺ (whereinW is a C₁₋₄alkyl group), and the like. For therapeutic use, salts of thecompounds provided herein are contemplated as being pharmaceuticallyacceptable. However, salts of acids and bases that arenon-pharmaceutically acceptable may also find use, for example, in thepreparation or purification of a pharmaceutically acceptable compound.

The term “solvate” as used herein, refers to the physical association ofa compound provided herein with one or more solvent molecules, whetherorganic or inorganic. This physical association often includes hydrogenbonding. In certain instances, the solvate is capable of isolation, forexample, when one or more solvate molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Exemplary solvates includehydrates, ethanolates, and methanolates.

The term “monovalent pharmaceutically acceptable cation” as used hereinrefers to inorganic cations such as, but not limited to, alkaline metalions, e.g., Na⁺ and K⁺, as well as organic cations such as, but notlimited to, ammonium and substituted ammonium ions, e.g., NH₄ ⁺, NHMe₃⁺, NH₂Me₂ ⁺, NHMe₃ ⁺ and NMe₄ ⁺.

The term “divalent pharmaceutically acceptable cation” as used hereinrefers to inorganic cations such as, but not limited to, alkaline earthmetal cations, e.g., Ca²⁺ and Mg²⁺.

Examples of monovalent and divalent pharmaceutically acceptable cationsare discussed, e.g., in Berge et al. J. Pharm. Sci., 66:1-19 (1997).

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent (including the compounds,pharmaceutical compositions, and compositions of matter provided herein)sufficient to result in amelioration of one or more symptoms of adisorder, or prevent advancement of a disorder, or cause regression ofthe disorder. For example, with respect to the treatment of cancer, inone embodiment, a therapeutically effective amount can refer to theamount of a therapeutic agent that decreases the rate of tumor growth,decreases tumor mass, decreases the number of metastases, increases timeto tumor progression, increase tumor cell apoptosis, or increasessurvival time by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

The terms “sensitize” and “sensitizing,” as used herein, refer tomaking, through the administration of a first therapeutic agent (e.g., acompound provided herein), an animal or a cell within an animal moresusceptible, or more responsive, to the biological effects (e.g.,promotion or retardation of an aspect of cellular function including,but not limited to, cell division, cell growth, proliferation, invasion,angiogenesis, necrosis, or apoptosis) of a second therapeutic agent. Thesensitizing effect of a first agent on a target cell can be measured asthe difference in the intended biological effect (e.g., promotion orretardation of an aspect of cellular function including, but not limitedto, cell growth, proliferation, invasion, angiogenesis, or apoptosis)observed upon the administration of a second agent with and withoutadministration of the first agent. The response of the sensitized cellcan be increased by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 100%, at least about 150%, at least about 200%, at least about250%, at least 300%, at least about 350%, at least about 400%, at leastabout 450%, or at least about 500% over the response in the absence ofthe first agent.

The term “dysregulation of apoptosis,” as used herein, refers to anyaberration in the ability of (e.g., predisposition) a cell to undergocell death via apoptosis. Dysregulation of apoptosis is associated withor induced by a variety of conditions, non-limiting examples of whichinclude, autoimmune disorders (e.g., systemic lupus erythematosus,rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, orSjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis,asthma or Crohn's disease), hyperproliferative disorders (e.g., tumors,B cell lymphomas, or T cell lymphomas), viral infections (e.g., herpes,papilloma, or HIV), and other conditions such as osteoarthritis andatherosclerosis. It should be noted that when the dysregulation isinduced by or associated with a viral infection, the viral infection mayor may not be detectable at the time dysregulation occurs or isobserved. That is, viral-induced dysregulation can occur even after thedisappearance of symptoms of viral infection.

The term “functional p53,” as used herein, refers to wild-type p53expressed at normal, high, or low levels and mutant or allelic variantsof p53 that retain(s) at least about 5% of the activity of wild-typep53, e.g., at least about 10%, about 20%, about 30%, about 40%, about50%, or more of wild-type activity.

The term “p53-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with p53, have tumor suppressoractivity, and are inhibited by interaction with MDM2 or MDM2-relatedproteins. Examples of p53-related proteins include, but are not limitedto, p63 and p73.

The term “MDM2-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with MDM2, and interact with andinhibit p53 or p53-related proteins. Examples of MDM2-related proteinsinclude, but are not limited to, MDMX and MDM4.

The term “senescence” as used herein, refers to the phenomenon wherebynon-cancerous diploid cells lose the ability to divide, andcharacterized in part by telomeric dysfunction or shortening.

The term “hyperproliferative disease,” as used herein, refers to anycondition in which a localized population of proliferating cells in ananimal is not governed by the usual limitations of normal growth.Examples of hyperproliferative disorders include tumors, neoplasms,lymphomas, leukemias and the like. A neoplasm is said to be benign if itdoes not undergo invasion or metastasis and malignant if it does eitherof these. A “metastatic” cell means that the cell can invade neighboringbody structures. Hyperplasia is a form of cell proliferation involvingan increase in cell number in a tissue or organ without significantalteration in structure or function. Metaplasia is a form of controlledcell growth in which one type of fully differentiated cell substitutesfor another type of differentiated cell.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjögren's syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, and the like.

The term “neoplastic disease,” as used herein, refers to any abnormalgrowth of cells being either benign (non-cancerous) or malignant(cancerous).

The term “melanoma” as used herein refers to any form of cancer thatbegins in melanocytes. Melanoma includes, but is not limited to, thefollowing subtypes: lentigo maligna, lentigo maligna melanoma,superficial spreading melanoma, acral lentiginous melanoma, mucosalmelanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma,amelanotic melanoma, soft-tissue melanoma, and metastatic melanoma.Melanoma, as used herein also includes metastatic melanoma.

The term “normal cell,” as used herein, refers to a cell that is notundergoing abnormal growth or division. Normal cells are non-cancerousand are not part of any hyperproliferative disease or disorder.

The term “anti-neoplastic agent,” as used herein, refers to any compoundthat retards the proliferation, growth, or spread of a targeted (e.g.,malignant) neoplasm.

The terms “prevent,” “preventing,” and “prevention,” as used herein,refer to a decrease in the occurrence of pathological cells (e.g.,hyperproliferative or neoplastic cells) in an animal. The prevention maybe complete, e.g., the total absence of pathological cells in a subject.The prevention may also be partial, such that the occurrence ofpathological cells in a subject is less than that which would haveoccurred without treatment with one or more compounds provided herein.

The terms “a” and “an” refer to one or more.

The term “apoptosis-modulating agents,” as used herein, refers to agentswhich are involved in modulating (e.g., inhibiting, decreasing,increasing, promoting) apoptosis. Examples of apoptosis-modulatingagents include proteins which comprise a death domain such as, but notlimited to, Fas/CD95, TRAMP, TNF RI, DR¹, DR², DR³, DR⁴, DR⁵, DR⁶, FADD,and RIP. Other examples of apoptosis-modulating agents include, but arenot limited to, TNFα, Fas ligand, antibodies to Fas/CD95 and other TNFfamily receptors, TRAIL (also known as Apo2 Ligand or Apo2L/TRAIL),antibodies to TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3kinase, PP1, and caspase proteins. Modulating agents broadly includeagonists and antagonists of TNF family receptors and TNF family ligands.Apoptosis-modulating agents may be soluble or membrane bound (e.g.ligand or receptor). Apoptosis-modulating agents include those which areinducers of apoptosis, such as TNF or a TNF-related ligand, particularlya TRAMP ligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” encompasses any of the standard pharmaceuticalcarriers, solvents, surfactants, or vehicles. Suitable pharmaceuticallyacceptable vehicles include aqueous vehicles and nonaqueous vehicles.Standard pharmaceutical carriers and their formulations are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,19th ed. 1995.

The term “alkyl” as used herein by itself or part of another grouprefers to a straight-chain or branched saturated aliphatic hydrocarbonhaving from one to eighteen carbons or the number of carbons designated(e.g., C₁-C₁₈ means 1 to 18 carbons). In one embodiment, the alkyl is aC₁-C₁₀ alkyl. In another embodiment, the alkyl is a C₄-C₈ alkyl. Inanother embodiment, the alkyl is a C₁-C₆ alkyl. In another embodiment,the alkyl is a C₁-C₄ alkyl. Exemplary alkyl groups include methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, n-hexyl, isohexyl, n-heptyl, 4,4-dimethylpentyl, n-octyl,2,2,4-trimethylpentyl, nonyl, decyl and the like.

The term “optionally substituted alkyl” as used herein by itself or partof another group means that the alkyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from hydroxy (i.e., —OH), nitro (i.e., —NO₂),cyano (i.e., —CN), optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. In oneembodiment, the optionally substituted alkyl is substituted with twosubstituents. In another embodiment, the optionally substituted alkyl issubstituted with one substituent. In another embodiment, thesubstituents are selected from hydroxyl (i.e., a hydroxyalkyl),optionally substituted cycloalkyl (i.e., a (cycloalkyl)alkyl), or amino(i.e., an aminoalkyl). Exemplary optionally substituted alkyl groupsinclude —CH₂OCH₃, —CH₂CH₂NH₂, —CH₂CH₂NH(CH₃), —CH₂CH₂CN, —CH₂SO₂CH₃,hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

The term “alkylenyl” as used herein by itself or part of another grouprefers to a divalent alkyl radical containing one, two, three, four, ormore joined methylene groups. Exemplary alkylenyl groups include—(CH₂)—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, and the like.

The term “optionally substituted alkylenyl” as used herein by itself orpart of another group means the alkylenyl as defined above is eitherunsubstituted or substituted with one, two, three, or four substituentsindependently selected from the group consisting of optionallysubstituted C₁-C₆ alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl. In oneembodiment, the optionally substituted C₁-C₆ alkyl is methyl. In oneembodiment, the optionally substituted aryl is a phenyl optionallysubstituted with one or two halo groups. Exemplary optionallysubstituted alkylenyl groups include —CH(CH₃)—, —C(CH₃)₂—, —CH₂CH(CH₃)—,—CH₂CH(CH₃)CH₂—, —CH₂CH(Ph)CH₂—, —CH(CH₃)CH(CH₃)—, and the like.

The term “haloalkyl” as used herein by itself or part of another grouprefers to an alkyl as defined above having one to six halo substituents.In one embodiment, the haloalkyl has one, two or three halosubstituents. Exemplary haloalkyl groups include trifluoromethyl,—CH₂CH₂F and the like.

The term “hydroxyalkyl” as used herein by itself or part of anothergroup refers to an alkyl as defined above having one hydroxysubstituent. Exemplary hydroxyalkyl groups include hydroxymethyl,hydroxyethyl, hydroxypropyl, and the like.

The term “dihydroxyalkyl” as used herein by itself or part of anothergroup refers to alkyl as defined above having two hydroxyl substituents.Exemplary dihydroxyalkyl groups include —CH₂CH₂CCH₃(OH)CH₂OH,—CH₂CH₂CH(OH)CH(CH3)OH, —CH₂CH(CH₂OH)₂,—CH₂CH₂CH(OH)C(CH₃)₂OH—CH₂CH₂CCH₃(OH)CH(CH₃)OH, and the like, includingstereoisomers thereof.

The term “hydroxycycloalkyl” as used herein by itself or part of anothergroup refers to an optionally substituted cycloalkyl as defined belowhaving a least one, e.g., one or two, hydroxy substituents. Exemplaryhydroxycycloalkyl groups include:

and the like, including stereoisomers thereof.

The term “optionally substituted (cycloalkyl)alkyl” as used herein byitself or part of another group refers to an optionally substitutedalkyl as defined above having an optionally substituted cycloalkyl (asdefined below) substituent. Exemplary optionally substituted(cycloalkyl)alkyl groups include:

and the like, including stereoisomers thereof.

The term “aralkyl” as used herein by itself or part of another grouprefers to an optionally substituted alkyl as defined above having one,two or three optionally substituted aryl substituents. In oneembodiment, the aralkyl has two optionally substituted arylsubstituents. In another embodiment, the aralkyl has one optionallysubstituted aryl substituent. In another embodiment, the aralkyl is anaryl(C₁-C₄ alkyl). In another embodiment, the aryl(C₁-C₄ alkyl) has twooptionally substituted aryl substituents. In another embodiment, thearyl(C₁-C₄ alkyl) has one optionally substituted aryl substituent.Exemplary aralkyl groups include, for example, benzyl, phenylethyl,(4-fluorophenyl)ethyl, phenylpropyl, diphenylmethyl (i.e., Ph₂CH—),diphenylethyl (Ph₂CHCH₂—) and the like.

The term “cycloalkyl” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic hydrocarbon groups containing one to three ringshaving from three to twelve carbon atoms (i.e., C₃-C₁₂ cycloalkyl) orthe number of carbons designated. In one embodiment, the cycloalkyl hasone ring. In another embodiment, the cycloalkyl is a C₃-C₆ cycloalkyl.Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin,adamantyl and the like.

The term “optionally substituted cycloalkyl” as used herein by itself orpart of another group means the cycloalkyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,aralkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclo,alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Theterm “optionally substituted cycloalkyl” also means the cycloalkyl asdefined above may be fused to an optionally substituted aryl. Exemplaryoptionally substituted cycloalkyl groups include:

and the like.

The term “alkenyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one, two or threecarbon-to-carbon double bonds. In one embodiment, the alkenyl has onecarbon-to-carbon double bond. Exemplary alkenyl groups include —CH═CH₂,—CH₂CH═CH₂, —CH₂CH₂CH═CH₂, —CH₂CH₂CH═CHCH₃ and the like.

The term “optionally substituted alkenyl” as used herein by itself orpart of another group means the alkenyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplaryoptionally substituted alkenyl groups include —CH═CHPh, —CH₂CH═CHPh andthe like.

The term “cycloalkenyl” as used herein by itself or part of anothergroup refers to a cycloalkyl group as defined above containing one, twoor three carbon-to-carbon double bonds. In one embodiment, thecycloalkenyl has one carbon-to-carbon double bond. Exemplarycycloalkenyl groups include cyclopentene, cyclohexene and the like.

The term “optionally substituted cycloalkenyl” as used herein by itselfor part of another group means the cycloalkenyl as defined above iseither unsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido.

The term “alkynyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one to threecarbon-to-carbon triple bonds. In one embodiment, the alkynyl has onecarbon-to-carbon triple bond. Exemplary alkynyl groups include —C≡CH,—C≡CCH₃, —CH₂C≡CH, —CH₂CH₂C≡CH and —CH₂CH₂C≡CCH₃.

The term “optionally substituted alkynyl” as used herein by itself orpart of another group means the alkynyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplaryoptionally substituted alkenyl groups include —C≡CPh, —CH₂C≡CPh and thelike.

The term “aryl” as used herein by itself or part of another group refersto monocyclic and bicyclic aromatic ring systems having from six tofourteen carbon atoms (i.e., C₆-C₁₄ aryl) such as phenyl (abbreviated asPh), 1-naphthyl and 2-naphthyl and the like.

The term “optionally substituted aryl” as used herein by itself or partof another group means the aryl as defined above is either unsubstitutedor substituted with one to five substituents independently selected fromhalo, nitro, cyano, hydroxy, amino, optionally substituted alkyl,haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy,alkylthio, carboxamido or sulfonamido. In one embodiment, the optionallysubstituted aryl is an optionally substituted phenyl. In one embodiment,the optionally substituted phenyl has four substituents. In anotherembodiment, the optionally substituted phenyl has three substituents. Inanother embodiment, the optionally substituted phenyl has twosubstituents. In another embodiment, the optionally substituted phenylhas one substituent. Exemplary substituted aryl groups include2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl,2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl,3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl,4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl,2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl,3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl3,5-di-methylphenyl and 3,5-dimethoxy, 4-methylphenyl,2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl and the like. The termoptionally substituted aryl is meant to include groups having fusedoptionally substituted cycloalkyl and fused optionally substitutedheterocyclo rings. Examples include:

and the like.

The term “heteroaryl” as used herein by itself or part of another grouprefers to monocyclic and bicyclic aromatic ring systems having from fiveto fourteen carbon atoms (i.e., C₅-C₁₄ heteroaryl) and one, two, threeor four heteroatoms independently selected from the group consisting ofoxygen, nitrogen and sulfur. In one embodiment, the heteroaryl has threeheteroatoms. In one embodiment, the heteroaryl has two heteroatoms. Inone embodiment, the heteroaryl has one heteroatom. Exemplary heteroarylgroups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, purinyl,2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 2-benzthiazolyl,4-benzthiazolyl, 5-benzthiazolyl, 5-indolyl, 3-indazolyl, 4-indazolyl,5-indazolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl,5-quinoxalinyl, 2-quinolyl 3-quinolyl, 6-quinolyl and the like. The termheteroaryl is meant to include possible N-oxides. Exemplary N-oxidesinclude pyridyl N-oxide and the like.

The term “optionally substituted heteroaryl” as used herein by itself orpart of another group means the heteroaryl as defined above is eitherunsubstituted or substituted with one to four substituents, typicallyone or two substituents, independently selected from halo, nitro, cyano,hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl,aralkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclo,alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Inone embodiment, the optionally substituted heteroaryl has onesubstituent. In another embodiment, the substituent is an optionallysubstituted aryl, aralkyl, or optionally substituted alkyl. In anotherembodiment, the substituent is an optionally substituted phenyl. Anyavailable carbon or nitrogen atom may be substituted. Exemplaryoptionally substituted heteroaryl groups include:

and the like.

The term “heterocyclo” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic groups containing one to three rings having fromtwo to twelve carbon atoms (i.e., C₂-C₁₂ heterocyclo) and one or twooxygen, sulfur or nitrogen atoms. The heterocyclo can be optionallylinked to the rest of the molecule through a carbon or nitrogen atom.Exemplary heterocyclo groups include:

and the like.

The term “optionally substituted heterocyclo” as used herein by itselfor part of another group means the heterocyclo as defined above iseither unsubstituted or substituted with one to four substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c),—SO₂R^(d), —N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e))C═N(R^(h))-amino, wherein R^(c) is hydrogen, optionallysubstituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl; R^(d) is optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl; R^(e)is hydrogen, optionally substituted alkyl, optionally substituted aryl,or optionally substituted heteroaryl; R^(f) is hydrogen, optionallysubstituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl; R^(g) is optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl; andR^(h) is hydrogen, —CN, optionally substituted alkyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Substitution mayoccur on any available carbon or nitrogen atom. Exemplary substitutedheterocyclo groups include:

and the like. An optionally substituted heterocyclo may be fused to anaryl group to provide an optionally substituted aryl as described above.

The term “alkoxy” as used herein by itself or part of another grouprefers to a haloalkyl, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl or optionallysubstituted alkynyl attached to a terminal oxygen atom. Exemplary alkoxygroups include methoxy, tert-butoxy, —OCH₂CH═CH₂ and the like.

The term “aryloxy” as used herein by itself or part of another grouprefers to an optionally substituted aryl attached to a terminal oxygenatom. Exemplary aryloxy groups include phenoxy and the like.

The term “aralkyloxy” as used herein by itself or part of another grouprefers to an aralkyl attached to a terminal oxygen atom. Exemplaryaralkyloxy groups include benzyloxy and the like.

The term “alkylthio” as used herein by itself or part of another grouprefers to a haloalkyl, aralkyl, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl or optionallysubstituted alkynyl attached to a terminal sulfur atom. Exemplary alkylgroups include —SCH₃ and the like.

The term “halo” or “halogen” as used herein by itself or part of anothergroup refers to fluoro, chloro, bromo or iodo. In one embodiment, thehalo is fluoro or chloro.

The term “amino” as used herein by itself or part of another grouprefers to a radical of formula —NR^(a)R^(b) wherein R^(a) and R^(b) areindependently hydrogen, haloalkyl, aralkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedheterocyclo, optionally substituted aryl or optionally substitutedheteroaryl; or R^(a) and R^(b) taken together with the nitrogen atom towhich they are attached form a four to seven membered optionallysubstituted heterocyclo. Exemplary amino groups include —NH₂, —N(H)CH₃,—N(CH₃)₂, N(H)CH₂CH₃, N(CH₂CH₃), —N(H)CH₂Ph and the like.

The term “carboxamido” as used herein by itself or part of another grouprefers to a radical of formula —CO-amino. Exemplary carboxamido groupsinclude —CONH₂, —CON(H)CH₃, —CON(H)Ph, —CON(H)CH₂CH₂Ph, —CON(CH₃)₂,CON(H)CHPh₂ and the like.

The term “sulfonamido” as used herein by itself or part of another grouprefers to a radical of formula —SO₂-amino. Exemplary sulfonamido groupsinclude —SO₂NH₂, —SO₂N(H)CH₃, —SO₂N(H)Ph and the like.

The term “about,” as used herein, includes the recited number ±10%.Thus, “about 10” means 9 to 11.

Certain of the compounds of the present disclosure may exist asstereoisomers, i.e., isomers that differ only in the spatial arrangementof atoms, including optical isomers and conformational isomers (orconformers). The disclosure includes all stereoisomers, both as pureindividual stereoisomer preparations and enriched preparations of each,and both the racemic mixtures of such stereoisomers as well as theindividual diastereomers and enantiomers that may be separated accordingto methods that are well known to those of skill in the art.

The term “substantially free of” as used herein means that the compoundcomprises less than about 25% of other stereoisomers, e.g.,diastereomers and/or enantiomers, as established using conventionalanalytical methods routinely used by those of skill in the art. In someembodiments, the amount of other stereoisomers is less than about 24%,less than about 23%, less than about 22%, less than about 21%, less thanabout 20%, less than about 19%, less than about 18%, less than about17%, less than about 16%, less than about 15%, less than about 14%, lessthan about 13%, less than about 12%, less than about 11%, less thanabout 10%, less than about 9%, less than about 8%, less than about 7%,less than about 6%, less than about 5%, less than about 4%, less thanabout 3%, less than about 2%, less than about 1%, or less than about0.5%.

Stereoisomerically enriched compounds that contain about 95% or more ofa desired stereoisomer, for example, about 96% or more, about 97% ormore, about 98% or more, or about 99% or more are referred to herein as“substantially pure” or “substantially pure stereoisomers.”

Stereoisomerically enriched compounds that contain about 99% or more ofa desired stereoisomer are referred to herein as “pure” or “purestereoisomers.” The purity of any stereoisomerically enriched compoundcan be determined using conventional analytical methods such as, forexample, normal phase HPLC, reverse phase HPLC, chiral HPLC, and ¹H and¹³C NMR.

Compounds

In certain embodiments, compounds of Formula I are provided:

wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are independently selected from thegroup consisting of hydrogen, halogen, hydroxy, amino, nitro, cyano,alkoxy, aryloxy, optionally substituted alkyl, haloalkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl, optionallysubstituted cycloalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, carboxamido, and sulfonamido;

R² is selected from the group consisting of optionally substituted aryl,aralkyl, and optionally substituted heteroaryl;

R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedcycloalkenyl, optionally substituted aryl, and optionally substitutedheteroaryl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R⁵ is selected from the group consisting of:

wherein:

each R^(6a) and R^(6b) is independently selected from the groupconsisting of hydrogen and optionally substituted C₁-C₆ alkyl;

R⁷ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(8a) and R^(8b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₆ alkyl, andoptionally substituted cycloalkyl; or

R^(8a) and R^(8b) taken together with the carbon that they are attachedform a 3- to 8-membered optionally substituted cycloalkyl;

W¹ is selected from the group consisting of —OR^(9a) and —NR^(9b)R^(9c);

R^(9a) is hydrogen;

R^(9b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(9d), and—CONR^(9e)R^(9f);

R^(9c) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(9b) and R^(9c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(9d) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(9e) and R^(9f) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(9e) and R^(9f) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);with the proviso that when W¹ is —OR^(9a) and W² is —OR¹⁰ then at leastone of R⁷, R^(8a), and R^(8b) is other than hydrogen;

R¹⁰ is hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) together with the nitrogen atom to which they areattached form a 4- to 8-membered optionally substituted heterocyclo;

n is 1, 2, 3, 4, or 5;

each R^(12a), R^(12b), R^(12c) and R^(12d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁴ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of —OR¹⁵ and —NR^(16a)R^(16b);or

Z and R¹⁴ taken together form a carbonyl, i.e., a C═O, group.

R¹⁵ is selected from the group consisting of hydrogen and metabolicallycleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

o is 1, 2, or 3;

p is 0, 1, 2, or 3;

each R^(17a), R^(17b), R^(17c) and R^(17d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹⁸ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

q is 0, 1, 2, or 3;

r is 1, 2, or 3;

each R^(22a), R^(22b), R^(22c), and R^(22d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R²³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R²⁴ is selected from the group consisting of —SO₂R^(24a) and—CONR^(24b)R^(24c);

R^(24a) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(24b) and R^(24c) are each independently selected from the groupconsisting of hydrogen, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(24b) and R^(24c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

s and t are each independently 1, 2, or 3;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, and optionally substituted cycloalkyl;

R″ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, optionally substituted cycloalkyl, and—COR³¹;

R³¹ is selected from the group consisting of hydrogen and optionallysubstituted alkyl; and

represents a single or a double bond,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, for the compounds of Formula I,

represents a single bond.

In certain embodiments, the compound of Formula I is a mixture ofstereoisomers, e.g., a mixture of diastereomers and/or enantiomers,e.g., a racemic mixture. In another such embodiment, the compound is amixture of diastereomers. In another such embodiment, the compound is amixture of enantiomers. In particular embodiments, the compound is asingle enantiomer.

In certain embodiments, R⁵ is selected from the group consisting of R5-1and R5-2. In particular embodiments, R⁵ is R5-2 and Z is —OH. Inparticular embodiments, R⁵ is R5-2A and Z is OH.

In certain embodiments, compounds of Formula Ia are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y havethe meanings as described above for Formula I, or pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula Ib are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y havethe meanings as described above for Formula I, or tautomer thereof, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula II-XVII are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y havethe meanings as described above for Formula I, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula II are provided, whereinR^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y have themeanings as described above in connection with Formula I, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula II are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae III-XVII.In some embodiments, compounds of Formula II are substantially purestereoisomers. In some embodiments, compounds of Formula II are purestereoisomers.

In some embodiments, compounds of Formula XIII are substantially free ofone or more other stereoisomers. In some embodiments, compounds ofFormula XIII are substantially pure stereoisomers. In some embodiments,compounds of Formula XIII are pure stereoisomers.

In some embodiments, compounds of Formula XIV are substantially free ofone or more other stereoisomers. In some embodiments, compounds ofFormula XIV are substantially pure stereoisomers. In some embodiments,compounds of Formula XIV are pure stereoisomers.

In some embodiments, compounds of Formula VI are provided, whereinR^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y have themeanings as described above in connection with Formula I, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula VI are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae II-V andVII-XVII. In some embodiments, compounds of Formula VI are substantiallypure stereoisomers. In some embodiments, compounds of Formulae VI arepure stereoisomers.

In some embodiments, compounds of Formula X are provided, whereinR^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y have themeanings as described above in connection with Formula I, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula X are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae II-IX andXI-XVII. In some embodiments, compounds of Formula X are substantiallypure stereoisomers. In some embodiments, compounds of Formula X are purestereoisomers.

In some embodiments, compounds of Formula XII are provided, whereinR^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y have themeanings as described above in connection with Formula I, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula XII are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae II-XI andXIII-XVII. In some embodiments, compounds of Formula XII aresubstantially pure stereoisomers. In some embodiments, compounds of XIIare pure stereoisomers.

In some embodiments, compounds of Formula XII are unexpectedly morepotent than compounds of Formulae II-XI and XIII-XVII. For example, asdemonstrated herein, compounds of Formula XII have lower IC₅₀ valuesthan compounds of Formulae II-XI and XIII-XVII against MDM2. In someembodiments, compounds of Formula XII are about 2-fold or more, e.g.,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold,about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold,about 25-fold, about 30-fold, about 35-fold, about 40-fold, about45-fold, about 50-fold, or more, more potent than compounds of FormulaII in fluorescence polarization-based MDM2 binding assays. In someembodiments, compounds of Formula XII are unexpectedly more efficaciousthan compounds of Formulae II-XI and XIII-XVII in xenograft tumor modelsin mice and/or in other in vivo efficacy models.

In certain embodiments, compounds of Formulae I-XVII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided. In some embodiments, compounds of Formulae II, VI, X, and XII,or a pharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein:

a) R^(1a), R^(1b), R^(1c), and R^(1d) are independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

b) R^(1a) and R^(1d) are hydrogen; R^(1b) is selected from the groupconsisting of hydrogen and fluoro; and R^(1c) is selected from the groupconsisting of fluoro and chloro;

c) R² is optionally substituted phenyl;

d) R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, and optionallysubstituted cycloalkyl;

e) R⁴ is hydrogen;

f) X is NH;

g) X is O;

h) X is S;

i) Y is O;

j) Y is S;

k) Y is NH; or

X and Y are NH;

or any combination thereof.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-1; R^(6a) and R^(6b) are hydrogen; R⁷ isC₁-C₄ alkyl; R^(8a) and R^(8b) are hydrogen; W is —OR¹⁰, R⁹ and R¹⁰ arehydrogen; and n is 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-1; R^(6a) and R^(6b) are hydrogen; R⁷ isC₁-C₄ alkyl; R^(8a) and R^(8b) are hydrogen; W is —NR^(11a)R^(11b), R⁹is hydrogen; and n is 2.

In certain embodiments, the compounds of Formulae II, VI, X, and XII ora pharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-1; R^(6a) and R^(6b) are hydrogen; R⁷ isC₁-C₄ alkyl; R^(8a) and R^(8b) are hydrogen; W is —OR¹⁰, one of R⁹ andR¹⁰ is hydrogen and the other is a metabolically cleavable group; and nis 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-2; R^(12a), R^(12b), R^(12c), and R^(12d) areeach hydrogen; R¹³ is hydrogen; Z is —OR¹⁵ and R¹⁵ is hydrogen; o is 1or 2; and p is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-2; R^(12a), R^(12b), R^(12c), and R^(12d) areeach hydrogen; R¹³ is hydrogen; Z is —NR^(16a)R^(16b); o is 1 or 2; andp is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-2; R^(12a), R^(12b), R^(12c), and R^(12d) areeach hydrogen; R¹³ is hydrogen; Z is —OR¹⁵ and R¹⁵ a metabolicallycleavable group; o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-3; R^(17a), R^(17b), R^(17c), and R^(17d) areeach hydrogen; R¹⁸, R¹⁹, and R²⁰ are hydrogen; R^(21a) and R^(21b) arehydrogen; and q and r are 1.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-3; R^(17a), R^(17b), R^(17c), and R^(17d) areeach hydrogen; R¹⁸, R¹⁹, and R²⁰ are hydrogen; one of R^(21a) andR^(21b) is hydrogen and the other is a metabolically cleavable group;and q and r are 1.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-2A.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R² is selected from the group consisting of aralkyland optionally substituted aryl having the Formula R2-1:

and R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are eachindependently selected from the group consisting of hydrogen, halogen,hydroxy, nitro, amino, cyano, alkoxy, optionally substituted alkyl,haloalkyl, optionally substituted aryl, and optionally substitutedheteroaryl. In particular embodiments, R^(25a) is selected from thegroup consisting of hydrogen and fluoro; R^(25b) is chloro; R^(25c) isselected from the group consisting of hydrogen and fluoro; and R^(25d)and R^(25e) are hydrogen.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

including stereoisomers, e.g., enantiomers, thereof, wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(9b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(9d), and—CONR^(9e)R^(9f);

R^(9c) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(9b) and R^(9c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(9d) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(9e) and R^(9f) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(9e) and R^(9f) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, C₁-C₄ alkyl, orC₃-C₆ cycloalkyl;

R¹⁵ is hydrogen or a metabolically cleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

R²⁴ is selected from the group consisting of —SO₂R^(24a) and—CONR^(24b)R^(24c);

R^(24a) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl; and

R^(24b) and R^(24c) are each independently selected from the groupconsisting of hydrogen, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl, or

R^(24b) and R^(24c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo.

In certain embodiments, R⁵ is selected from the group consisting ofR5-5, R5-6, R5-10, R5-11, R5-12, R5-13, and R5-14.

In certain embodiments, R⁵ is selected from the group consisting ofR5-10 and R5-12 and R¹⁴ is hydrogen or methyl and R¹⁵ is hydrogen.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(8a) and R^(8b) are each independently selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl;

R^(8a) and R^(8b) are each independently is selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl;

R^(9d) is selected from the group consisting of methyl, trifluoromethyl,ethyl, propyl, isopropyl, and cyclopropyl; and

R^(11c) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl;

R^(8a) and R^(8b) are each independently is selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl;

R^(9e) is selected from the group consisting of methyl, trifluoromethyl,ethyl, propyl, isopropyl, and cyclopropyl; and

R^(11d) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R¹⁹ and R²⁰ are each independently is selected from the group consistingof hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(16c) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(16d) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formulae II, VI, X, and XII, or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is:

In another embodiment, compounds of Formula XVIIIa are provided:

wherein:

R^(1b) and R^(1c) are independently selected from the group consistingof hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, and, optionallysubstituted aryl, optionally substituted cycloalkyl;

R^(26a), R^(26b), and R^(26c) are independently selected from the groupconsisting of hydrogen, fluoro, and chloro; and

R²⁷ is selected from the group consisting of:

wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₄ alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of —OR¹⁵ and —NR^(16a)R^(16b);

R¹⁵ is selected from the group consisting of hydrogen and metabolicallycleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIb are provided:

wherein R^(1b), R^(1b), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above for Formula XVIIIa, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIc are provided:

wherein R^(1b), R^(1b), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above for Formula XVIIIa, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In certain embodiments, R²⁷ is selected from the group consisting ofR27-2, R27-3, R27-5, R27-6, R27-8, R27-9, R27-11, R27-12, R27-14,R27-15, R27-16, R27-17, R27-19, R27-20, R27-21, R27-22, R27-24, R27-25,R27-27, R27-29, R27-30, R27-31, and R27-32. In certain embodiments, R²⁷is selected from the group consisting of R27-2, R27-3, R27-5, and R27-6,R27-8, R27-9, R27-14, R27-15, R27-16, and R27-17. In certainembodiments, R²⁷ is a hydroxycycloalkyl group.

In certain embodiments, R^(9a) is hydrogen; W² is OH; Z is OH; R⁷ isC₁-C₄ alkyl, e.g., methyl, ethyl, propyl, or isopropyl, or cyclopropyl;R¹⁴, R¹⁹, and R²⁰ are each independently hydrogen, C₁-C₄ alkyl, e.g.,methyl, ethyl, propyl, or isopropyl, or cyclopropyl; and R^(21a) andR^(21b) are each hydrogen.

In certain embodiments, R^(9a) is hydrogen, R⁷ is hydrogen, C₁-C₄ alkyl,or cyclopropyl; W² is —NHR^(11a); R^(11a) is C₁-C₄ alkyl, e.g., methyl,trifluoromethyl, ethyl, propyl, or isopropyl, or cyclopropyl; R¹⁴ ishydrogen, C₁-C₄ alkyl, e.g., methyl, ethyl, propyl, or isopropyl, orcyclopropyl; Z is —NHSO₂R^(16c) or —NHCONHR^(16d); and R^(16c) andR^(16d) are each independently optionally substituted C₁-C₄ alkyl, e.g.,methyl, trifluoromethyl, ethyl, propyl, or isopropyl, or cyclopropyl.

In certain embodiments, compounds of Formulae XIX-XXXIV are provided:

wherein R^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, whereinR^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have the meaningsas described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula XIX are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae XX-XXXIV.In some embodiments, compounds of Formula XIX are substantially purestereoisomers. In some embodiments, compounds of Formula XIX are purestereoisomers.

In certain embodiments, compounds of Formula XXIII are provided, whereinR^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have the meaningsas described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula XXIII are substantially freeof one or more other stereoisomers, i.e., compounds of Formulae XIX-XXIIand XXIV-XXXIV. In some embodiments, compounds of Formula XXIII aresubstantially pure stereoisomers. In some embodiments, compounds ofFormula XXIII are pure stereoisomers.

In certain embodiments, compounds of Formula XXVII are provided, whereinR^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have the meaningsas described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula XXVII are substantially freeof one or more other stereoisomers, i.e., compounds of Formulae XIX-XXVIand XXVIII-XXXIV. In some embodiments, compounds of Formula XXVII aresubstantially pure stereoisomers. In some embodiments, compounds ofFormula XXVII are pure stereoisomers.

In certain embodiments, compounds of Formula XXIX are provided, whereinR^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have the meaningsas described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula XXIX are substantially free ofone or more other stereoisomers, i.e., compounds of Formulae XIX-XXVIIIand XXX-XXIV. In some embodiments, compounds of Formula XXIX aresubstantially pure stereoisomers. In some embodiments, compounds ofFormula XXIX are pure stereoisomers.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, andXXIX are provided, wherein R²⁷ is selected from the group consisting of:

wherein:

R⁷ is C₁-C₄ alkyl;

R^(9a) and R¹⁰ are hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is metabolicallycleavable group;

R^(11a) and R^(11b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₄ alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen and C₁-C₄ alkyl;

R¹⁵ is hydrogen or a metabolically cleavable group; and

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16c) is selected from the group consisting of optionally substitutedC₁-C₄ alkyl or cyclopropyl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₄ alkyl orcyclopropyl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, andXXIX are provided, wherein R²⁷ is selected from the group consisting of:

wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, andXXIX are provided, wherein R²⁷ is selected from the group consisting of:

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, andXXIX are provided, wherein R²⁷ is selected from the group consisting of:

wherein R^(11a) and R^(11b) taken together with the nitrogen to whichthey are attached form a 5- or 6-membered optionally substitutedheterocyclo.

In certain embodiments, compounds of Formulae XIX, XXIII, XXVII, andXXIX are provided, wherein R²⁷ is selected from the group consisting of:

wherein:R¹⁴ is selected from the group consisting of hydrogen and C₁-C₄ alkyl;andR¹⁵ is a metabolically cleavable group.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX,XXIII, XXVII, and XXIX are provided, wherein R¹⁵ is a metabolicallycleavable group selected from the group consisting of:

wherein:

each R^(28a) and R^(28b) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and aralkyl;

R^(29a) and R^(29b) are each selected from the group consisting ofhydrogen and optionally substituted alkyl;

v is 1, 2, 3, or 4; and

R^(30a) and R^(30b) are each selected from the group consisting ofhydrogen, optionally substituted alkyl, aralkyl, optionally substitutedaryl, and monovalent pharmaceutically acceptable cation; or

taken together R^(30a) and R^(30b) represent a divalent pharmaceuticallyacceptable cation or an optionally substituted alkylenyl.

In certain embodiments, R¹⁵ is the residue of a natural or unnaturalamino acid. In other embodiments, R¹⁵ is the residue of glycine,isoleucine alanine, leucine, asparagine, lysine, aspartic acid,methionine, cysteine, phenylalanine, glutamic acid, threonine,glutamine, tryptophan, valine, proline, serine, tyrosine, arginine, andhistidine.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX,XXIII, XXVII, and XXIX are provided, wherein R³ is C₁-C₁₀ alkyl.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX,XXIII, XXVII, and XXIX are provided, wherein R³ is selected from thegroup consisting of —CH₂C(CH₃)₃, —CH₂C(CH₃)₂CH₂CH₃,—CH₂C(CH₃)₂CH₂CH₂CH₃, —CH₂C(CH₃)₂CH₂CH₂CH₂CH₃, —CH₂C(CH₂CH₃)₂CH₃, and—CH₂C(CH₃)₂CH₂CH(CH₃)₂. In certain embodiments, R³ is —CH₂C(CH₃)₃.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX,XXIII, XXVII, and XXIX are provided, wherein R³ is optionallysubstituted aryl.

In certain embodiments, compounds of Formulae II, VI, X, XII, XIX,XXIII, XXVII, and XXIX are provided, wherein R³ is optionallysubstituted phenyl.

In some embodiments, compounds of Formulae XIX, XXIII, XXVII, and XXIXare provided wherein R^(1b), R^(1c), R^(26a), R^(26b), and R^(26c) areeach independently selected from the group consisting of hydrogen andhalogen, e.g., chloro or fluoro, or a pharmaceutically acceptable salt,solvate, or prodrug thereof.

In certain embodiments, compounds having Formulae VI, X, and XII areprovided, wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is selected from the group consisting of aralkyl and:

wherein:

R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted C₁-C₈alkyl and optionally substituted aryl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; and

R″ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₄ alkyl, and —COCH₃,

wherein the compound is substantially free of one or more otherstereoisomers,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, R⁴ is hydrogen. In some embodiments, X is NH. Insome embodiments, Y is NH. In some embodiments, R³ is —CH₂C(CH₃)₃. Insome embodiments, R⁵ is selected from the group consisting of:

In certain embodiments, compounds having Formula XII are provided,wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is selected from the group consisting of benzyl and:

wherein:

R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substituted C₁-C₈alkyl and phenyl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; and

R″ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₄ alkyl, and —COCH₃,

wherein the compound is substantially free of one or more otherstereoisomers,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, R⁴ is hydrogen. In some embodiments, X is NH. Insome embodiments, Y is NH. In some embodiments, R³ is —CH₂C(CH₃)₃. Insome embodiments, R⁵ is selected from the group consisting of:

In certain embodiments, compounds having Formula XII are provided,wherein:

R^(1a) is hydrogen;R^(1b), R^(1c), and R^(1d) are each independently selected from thegroup consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;R³ is C₄-C₈ alkyl;R⁴ is hydrogen;R⁵ is selected from the group consisting of:

and

X and Y are NH,

wherein the compound is substantially free of one or more otherstereoisomers,or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, R⁵ is selected from the group consisting of:

In some embodiments, compounds having Formula XXXV:

are provided, wherein:R^(1b) and R^(1c) are independently selected from the group consistingof hydrogen, fluoro, and chloro;R³ is C₄-C₈ alkyl; andR^(25a), R^(25b), and R^(25c) are each independently selected from thegroup consisting of hydrogen, fluoro, and chloro,or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds having Formula XXXV are substantiallyfree of one or more other stereoisomers. In some embodiments, compoundshaving Formula XXXV are substantially pure stereoisomers. In someembodiments, compounds having Formula XXXV are pure stereoisomers.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In some embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In some embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided,wherein the compound is substantially free of one or more otherstereoisomers.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided,wherein the compound is substantially free of one or more otherstereoisomers.

In certain embodiments, a compound having the following structure:

or a pharmaceutically acceptable salt or solvate thereof is provided,wherein the compound is a substantially pure stereoisomer.

In certain embodiments, a compound having the following structure:

or a pharmaceutically acceptable salt or solvate thereof is provided,wherein the compound is a pure stereoisomer.

In certain embodiments, methods of preparing a compound having FormulaXXXVII:

are provided, wherein:

R³² is selected from the group consisting of —OR³³ and —NR^(34a)R^(34b);

R³³ is selected from the group consisting of hydrogen, alkyl, andaralkyl;

R^(34a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, aralkyl,optionally substituted aryl, and optionally substituted heteroaryl;

R^(34b) is selected from the group consisting of hydrogen and alkyl;

R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is selected from the group consisting of aralkyl and:

R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro; and

R³ is selected from the group consisting of optionally substituted C₁-C₈alkyl and optionally substituted aryl.

In one embodiment, the method of preparing a compound having FormulaXXXVII comprises allowing a compound having Formula XXXVI:

to isomerize to a compound having Formula XXXVII:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, and R³² have themeanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having FormulaXXXVII comprises dissolving a compound having Formula XXXVI:

in a solvent or a mixture of solvents,wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, and R³² have themeanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having FormulaXXXVII comprises:

a) dissolving a compound having Formula XXXVI:

in a solvent or a mixture of solvents; and

b) allowing the compound having Formula XXXVI to isomerize to a compoundhaving Formula XXXVII,

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, and R³² have themeanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having FormulaXXXVII:

comprises:

a) allowing the compound having Formula XXXVI:

to isomerize to a compound having Formula XXXVII; and

b) isolating the compound having Formula XXXVII substantially free fromthe compound having Formula XXXVI, and one or more other stereoisomers,

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, and R³² have themeanings as described above in connection with Formula XXXVII.

In one embodiment, the method of preparing a compound having FormulaXXXVII:

comprises:

a) dissolving a compound having Formula XXXVI:

in a solvent or a mixture of solvents;

b) allowing the compound having Formula XXXVI to isomerize to a compoundhaving Formula XXXVII; and

c) isolating the compound having Formula XXXVII substantially free fromthe compound having Formula XXXVI, and one or more other stereoisomers,

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, and R³² have themeanings as described above in connection with Formula XXXVII.

In one embodiment, the solvent is selected from the group consisting ofacetonitrile, methanol, ethyl acetate, and water, or a mixture thereof.

In one embodiment, the isomerization is carried out at a pH of less than7, e.g., at a pH of about 6, about 5, about 4, about 3, about 2, orabout 1. In one embodiment, the isomerization is carried out at a pH ofabout 7. In one embodiment, the isomerization is carried out at a pH ofgreater than 7, e.g., at a pH of about 8, about 9, about 10, about 11,about 12, or about 13.

In one embodiment, the isomerization is carried out in the presence ofan acid, e.g., trifluoroacetic acid or acetic acid.

In one embodiment, the isomerization is carried out in the presence of abase, e.g., NaHCO₃.

In one embodiment, isomerization is carried out at a temperature ofabout 20° C. to about 100° C., e.g., at a temperature of about 25° C. toabout 70° C., e.g., at a temperature of about 45° C. to about 65° C. Inone embodiment the isomerization is carried out at about roomtemperature, e.g., at about 25° C. In one embodiment the isomerizationis carried out above room temperature, e.g., at about 30° C., about 35°C., about 40° C., about 45° C., about 50° C., about 55° C., about 60°C., about 65° C., about 70° C., about 75° C., about 80° C., about 85°C., about 90° C., about 95° C., or about 100° C.

In one embodiment, the isomerization is carried about for a period oftime between about 0.5 hours and about 2 weeks, e.g., for about 1 hour,about 3 hours, about 6 hours, about 12 hours, about 1 day, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week.The period of time needed for isomerization to occur may depend on avariety of factors including the chemical structure of Formula XXXVI,the solvent(s), the temperature, and/or the pH.

In one embodiment, R³² is —OR³³.

In one embodiment, R³² is —NR^(34a)R^(34b).

In one embodiment, R^(34b) is hydrogen and R^(34a) is selected from thegroup consisting of alkyl, hydroxyalkyl, hydroxycycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl.

In one embodiment, R^(34b) is hydrogen and R^(34a) is selected from thegroup consisting of:

In one embodiment, the compound having Formula XXXVII is isolated as asubstantially pure stereoisomer. In one embodiment, the compound havingFormula XXXVII is isolated as a pure stereoisomer.

In one embodiment,

R³² is —NR^(34a)R^(34b); R^(34a) is:

R^(34b) is hydrogen;

R² is:

andR³ is C₁-C₈ alkyl.

In one embodiment, a method of preparing MI-77301 comprising allowingMI-773 to isomerize to MI-77301 is provided.

In one embodiment, a method of preparing MI-77301 comprising:

a) allowing MI-773 to isomerize to MI-77301; andb) isolating MI-77301 substantially free from one or more otherstereoisomers,is provided.

In one embodiment, a method of preparing MI-77301 comprising:

a) dissolving MI-773 in a solvent or a mixture of solvents;b) allowing MI-773 to isomerize to MI-77301; andc) isolating MI-77301 substantially free from one or more otherstereoisomers,is provided.

The compounds and processes provided herein will be better understood inconnection with the following synthetic schemes which illustrate themethods by which the compounds provided herein may be prepared. Startingmaterials can be obtained from commercial sources or prepared bywell-established literature methods known to those of ordinary skill inthe art. It will be readily apparent to one of ordinary skill in the artthat the compounds defined above can be synthesized by substitution ofthe appropriate reagents and agents in the syntheses shown below.

Compounds of Formula Ia wherein Y is NH can be synthesized as describedin Schemes 2 and 3.

Compounds of Formula Ia can be separated by chiral resolution methodswell known in the art, e.g., chiral column chromatography, to givecompounds of Formulae II-XVII. Suitable chiral columns for use in chiralresolutions include, for example, Daicel CHIRALCEL®OD-H, DaicelCHIRALPAK® AD-H and Regis Technologies ULMO chiral columns. Other chiralresolution methods are also possible. Compounds of Formulae II-XVII canalso be prepared by asymmetric synthetic methods. For example, compoundsof Formula II, wherein Y is NH, can be synthesized by using a asymmetric1,3-dipolar cycloaddition as the key step as previously described (SeeU.S. Pat. Nos. 7,759,383 B2 and 7,737,174 B2, and Ding et al., J. Am.Chem. Soc. 127:10130-10131 (2005)) (Scheme 4).

Briefly, compound A reacts with aldehyde B to give C. Compound C reactswith aldehyde E and compound D to give F (a compound of Formula Iwherein R″ is aralkyl). Treatment of F with Pb(OAc)₄ or CAN gives thecompound of Formula II wherein Y is NH.

Compounds of Formula XII can be prepared via isomerization of compoundsof Formula II. Without intending to be bound by theory, theisomerization of a compound having Formula II to a compound havingFormula XII (and other isomers, including compounds having Formula VI)may involve formation of the imine intermediate shown in Scheme 5.Compounds of Formula XII may be less likely to isomerize, i.e., they maybe chemically more stable, than compounds of Formula II.

Methods

In some embodiments, compounds provided herein induce cell cycle arrestand/or apoptosis and also potentiate the induction of cell cycle arrestand/or apoptosis either alone or in response to additional apoptosisinduction signals. Therefore, it is contemplated that these compoundssensitize cells to induction of cell cycle arrest and/or apoptosis,including cells that are resistant to such inducing stimuli. Byinhibiting the interaction between p53 or p53-related proteins and MDM2or MDM2-related proteins, the compounds provided herein can be used toinduce apoptosis in any disorder that can be treated, ameliorated, orprevented by the induction of apoptosis. In one embodiment, theinhibitors can be used to induce apoptosis in cells comprisingfunctional p53 or p53-related proteins.

In another embodiment, the disclosure pertains to modulating apoptosiswith compounds provided herein in combination with one or moreadditional apoptosis-modulating agents. Examples of apoptosis-modulatingagents include, but are not limited to, Fas/CD95, TRAMP, TNF RI, DR1,DR2, DR3, DR4, DR5, DR6, FADD, RIP, TNFα, Fas ligand, TRAIL, antibodiesto TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase,PP1, and caspase proteins. Other agents involved in the initiation,decision and degradation phase of apoptosis are also included. Examplesof apoptosis-modulating agents include agents, the activity, presence,or change in concentration of which, can modulate apoptosis in asubject. Apoptosis-modulating agents include those which are inducers ofapoptosis, such as TNF or a TNF-related ligand, particularly a TRAMPligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

In some embodiments, the compounds, compositions, and methods providedherein, including the methods comprising pulsatile dose administration,are used to treat diseased cells, tissues, organs, or pathologicalconditions and/or disease states in an animal (e.g., a mammalian patientincluding, but not limited to, humans and veterinary animals). In thisregard, various diseases and pathologies are amenable to treatment orprophylaxis using the present methods and compositions. A non-limitingexemplary list of these diseases and conditions includes, but is notlimited to, breast cancer, prostate cancer, lymphoma, skin cancer,pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovariancancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia (CLL) including B-CLL, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, sarcoma such as liposarcoma, malignant fibroushistiocytoma, osteosarcoma, Ewing's sarcoma, leiomyosarcoma, andrhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcomas such as lipoma, and malignant Schwannoma, osteogenic sarcoma,primary macroglobulinemia, and retinoblastoma, and the like, T and Bcell mediated autoimmune diseases; inflammatory diseases; infections;hyperproliferative diseases; AIDS; degenerative conditions, vasculardiseases, and the like. In some embodiments, the cancer cells beingtreated are metastatic. In other embodiments, the cancer cells beingtreated are resistant to other anticancer agents.

In some embodiments, the compounds, compositions, and methods providedherein, including the methods comprising pulsatile dose administration,are used to treat, ameliorate, or prevent a cancer selected from thegroup consisting of melanoma, lung cancer, a sarcoma, colon cancer,prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomachcarcinoma, acute myeloid leukemia, a lymphoma, multiple myeloma, and aleukemia in a patient.

In some embodiments, the compounds, compositions, and methods providedherein, including the methods comprising pulsatile dose administration,are used to treat, ameliorate, or prevent melanoma in a patient.

In some embodiments, the compounds, compositions, and methods providedherein, including the methods comprising pulsatile dose administration,are used to treat, ameliorate, or prevent liposarcoma in a patient.

In some embodiments, the compounds, compositions, and methods providedherein, including the methods comprising pulsatile dose administration,are used to treat cancers that express functional or wild type p53 orp53-related proteins. In some embodiments, the compounds, compositions,and methods provided herein are used to treat cancers that expresselevated levels of MDM2 or MDM2-related proteins.

In some embodiments, the methods, compounds, and compositions providedherein, including the methods comprising pulsatile dose administration,can be used to treat a patient having a sarcoma, including, for example,liposarcoma, malignant fibrous histiocytoma, osteosarcoma, andrhabdomyosarcoma. In some embodiments, the methods, compounds, andcompositions provided herein, including the methods comprising pulsatiledose administration, can be used to treat a patient having a soft tissuetumor, including, for example, Ewing's sarcoma, leiomyosarcoma, lipoma,and malignant Schwannomas. In some embodiments, the methods, compounds,and compositions provided herein, including the methods comprisingpulsatile dose administration, can be used to treat a patient havinglung, breast, liver, or colon cancer. In some embodiments, the methods,compounds, and compositions provided herein, including the methodscomprising pulsatile dose administration, can be used to treat a patienthaving B-cell chronic lymphocytic leukemia and acute myeloid leukemia.

In some embodiments, infections suitable for treatment with thecompounds, compositions, and methods provided herein include, but arenot limited to, infections caused by viruses, bacteria, fungi,mycoplasma, prions, and the like.

In some embodiments, methods are provided, including the methodscomprising pulsatile dose administration, for administering an effectiveamount of a compound or composition provided herein and at least oneadditional therapeutic agent (including, but not limited to,chemotherapeutic antineoplastics, apoptosis-modulating agents,antimicrobials, antivirals, antifungals, and anti-inflammatory agents)and/or therapeutic technique (e.g., surgical intervention, and/orradiotherapies). In a particular embodiment, the additional therapeuticagent(s) is an anticancer agent.

A number of suitable therapeutic or anticancer agents are contemplatedfor use in the methods provided herein, including the methods comprisingpulsatile dose administration. Indeed, the methods provided herein caninclude but are not limited to, administration of numerous therapeuticagents such as: agents that induce apoptosis; polynucleotides (e.g.,anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes andantibodies); biological mimetics (e.g., gossypol or BH3 mimetics);agents that bind (e.g., oligomerize or complex) with a Bcl-2 familyprotein such as Bax; alkaloids; alkylating agents; antitumorantibiotics; antimetabolites; hormones; platinum compounds; monoclonalor polyclonal antibodies (e.g., antibodies conjugated with anticancerdrugs, toxins, defensins), toxins; radionuclides; biological responsemodifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g.,IL-2)); adoptive immunotherapy agents; hematopoietic growth factors;agents that induce tumor cell differentiation (e.g., all-trans-retinoicacid); gene therapy reagents (e.g., antisense therapy reagents andnucleotides); tumor vaccines; angiogenesis inhibitors; proteosomeinhibitors: NF-κB modulators; anti-CDK compounds; HDAC inhibitors; andthe like. Numerous other examples of therapeutic agents such aschemotherapeutic compounds and anticancer therapies suitable forco-administration with the disclosed compounds are known to thoseskilled in the art.

In certain embodiments, anticancer agents comprise agents that induce orstimulate apoptosis. Agents that induce or stimulate apoptosis include,for example, agents that interact with or modify DNA, such as byintercalating, cross-linking, alkylating, or otherwise damaging orchemically modifying DNA. Agents that induce apoptosis include, but arenot limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosisfactor (TNF)-related factors (e.g., TNF family receptor proteins, TNFfamily ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinaseinhibitors (e.g., epidermal growth factor receptor (EGFR) kinaseinhibitor. Additional anticancer agents include: vascular growth factorreceptor (VGFR) kinase inhibitor, fibroblast growth factor receptor(FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR)kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC));antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, andAVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen);anti-androgens (e.g., flutamide, bicalutamide, finasteride,aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2(COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, andnon-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs(e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasoneintensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON,ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL,prednisolone, prednisone, PRELONE, and TANDEARIL); and cancerchemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11,fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone,MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin,gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL);cellular signaling molecules; ceramides and cytokines; staurosporine,and the like.

In still other embodiments, the compositions and methods providedherein, including the methods comprising pulsatile dose administration,include one or more compounds provided herein and at least oneanti-hyperproliferative or antineoplastic agent selected from alkylatingagents, antimetabolites, and natural products (e.g., herbs and otherplant and/or animal derived compounds).

Alkylating agents suitable for use in the present compositions andmethods include, but are not limited to: 1) nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin);and chlorambucil); 2) ethylenimines and methylmelamines (e.g.,hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan);4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine(methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes(e.g., dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the presentcompositions and methods include, but are not limited to: 1) folic acidanalogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs(e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine(fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP),thioguanine (6-thioguanine; TG), and pentostatin (2′-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for usein the compositions and methods of the present disclosure include, butare not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB),vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide);3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g.,L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin(cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide): and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present disclosure. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 1 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

TABLE 1 Aldesleukin Proleukin (des-alanyl-1, serine-125 humaninterleukin-2) Alemtuzumab Campath (IgG1κ anti CD52 antibody)Alitretinoin Panretin (9-cis-retinoic acid) Allopurinol Zyloprim(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one monosodium salt)Altretamine Hexalen (N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine-2,4,6-triamine) Amifostine Ethyol (ethanethiol, 2-[(3-aminopropyl)amino]-,dihydrogen phosphate (ester)) Anastrozole Arimidex(1,3-Benzenediacetonitrile, a,a,a′,a′-tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl)) Arsenic trioxide Trisenox Asparaginase Elspar(L-asparagine amidohydrolase, type EC-2) BCG Live TICE BCG (lyophilizedpreparation of an attenuated strain of Mycobacterium bovis (BacillusCalmette-Gukin [BCG], substrain Montreal) bexarotene capsules Targretin(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- napthalenyl) ethenyl]benzoic acid) bexarotene gel Targretin Bleomycin Blenoxane (cytotoxicglycopeptide antibiotics produced by Streptomyces verticillus; bleomycinA₂ and bleomycin B₂) Capecitabine Xeloda(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine) CarboplatinParaplatin (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine BCNU, BiCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) Carmustine with Polifeprosan 20Implant Gliadel Wafer Celecoxib Celebrex (as4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol- 1-yl]benzenesulfonamide) Chlorambucil Leukeran(4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin Platinol(PtCl₂H₆N₂) Cladribine Leustatin, 2-CdA(2-chloro-2′-deoxy-b-D-adenosine) Cyclophosphamide Cytoxan, Neosar(2-[bis(2-chloroethyl)amino] tetrahydro-2H-13,2- oxazaphosphorine2-oxide monohydrate) Cytarabine Cytosar-U(1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅) cytarabine liposomal DepoCytDacarbazine DTIC-Dome(5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC))Dactinomycin, actinomycin D Cosmegen (actinomycin produced byStreptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfa Aranesp(recombinant peptide) daunorubicin liposomal DanuoXome((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride) Daunorubicin HCl,daunomycin Cerubidine ((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1-naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride)Denileukin diftitox Ontak (recombinant peptide) Dexrazoxane Zinecard((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6-piperazinedione) DocetaxelTaxotere ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester,13-ester with 5b-20-epoxy-12a,4,7b,10b,13a- hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate) Doxorubicin HCl Adriamycin, Rubex(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride) doxorubicinAdriamycin PFS Intravenous injection doxorubicin liposomal Doxildromostanolone propionate Dromostanolone(17b-Hydroxy-2a-methyl-5a-androstan-3-one propionate) dromostanolonepropionate Masterone injection Elliott's B Solution Elliott's B SolutionEpirubicin Ellence ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-arabino-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione hydrochloride) Epoetinalfa Epogen (recombinant peptide) Estramustine Emcyt(estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate, or estradiol 3-[bis(2- chloroethyl)carbamate]17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposidephosphate Etopophos (4′-Demethylepipodophyllotoxin9-[4,6-O-(R)-ethylidene- (beta)-D-glucopyranoside], 4′-(dihydrogenphosphate)) etoposide, VP-16 Vepesid (4′-demethylepipodophyllotoxin9-[4,6-0-(R)-ethylidene- (beta)-D-glucopyranoside]) Exemestane Aromasin(6-methylenandrosta-1,4-diene-3,17-dione) Filgrastim Neupogen(r-metHuG-CSF) floxuridine (intraarterial) FUDR(2′-deoxy-5-fluorouridine) Fludarabine Fludara (fluorinated nucleotideanalog of the antiviral agent vidarabine, 9-b-D-arabinofuranosyladenine(ara-A)) Fluorouracil, 5-FU Adrucil(5-fluoro-2,4(1H,3H)-pyrimidinedione) Fulvestrant Faslodex(7-alpha-[9-(4,4,5,5,5-penta fluoropentylsulphinyl)nonyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Gemcitabine Gemzar(2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b- isomer))Gemtuzumab Ozogamicin Mylotarg (anti-CD33 hP67.6) Goserelin acetateZoladex Implant Hydroxyurea Hydrea Ibritumomab Tiuxetan Zevalin(immunoconjugate resulting from a thiourea covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan[N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)-ethyl]glycine) Idarubicin Idamycin(5,12-Naphthacenedione, 9-acetyl-7-[(3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEX(3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) Imatinib Mesilate Gleevec(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate)Interferon alfa-2a Roferon-A (recombinant peptide) Interferon alfa-2bIntron A (Lyophilized (recombinant peptide) Betaseron) Irinotecan HClCamptosar((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′:6,7] indolizino[1,2-b] quinoline-3,14(4H,12H) dionehydrochloride trihydrate) Letrozole Femara(4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) LeucovorinWellcovorin, Leucovorin (L-Glutamic acid,N[4[[(2amino-5-formyl1,4,5,6,7,8-hexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl Ergamisol ((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b] thiazole monohydrochloride C₁₁H₁₂N₂S•HCl) Lomustine CeeNU(1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine, nitrogenmustard Mustargen (2-chloro-N-(2-chloroethyl)-N-methylethanaminehydrochloride) Megestrol acetate Megace17α(acetyloxy)-6-methylpregna-4,6-diene-3,20-dione Melphalan, L-PAMAlkeran (4-[bis(2-chloroethyl) amino]-L-phenylalanine) Mercaptopurine,6-MP Purinethol (1,7-dihydro-6H-purine-6-thione monohydrate) MesnaMesnex (sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid) MethoxsalenUvadex (9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Mitomycin CMutamycin mitomycin C Mitozytrex Mitotane Lysodren(1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) MitoxantroneNovantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione dihydrochloride)Nandrolone phenpropionate Durabolin-50 Nofetumomab Verluma OprelvekinNeumega (IL-11) Oxaliplatin Eloxatin(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] [oxalato(2-)- O,O′] platinum)Paclitaxel TAXOL (5β,20-Epoxy-1,2a,4,7β,10β,13a-hexahydroxytax-11-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia (phosphonic acid(3-amino-1-hydroxypropylidene) bis-, disodium salt, pentahydrate, (APD))Pegademase Adagen (Pegademase ((monomethoxypolyethylene glycolsuccinimidyl) 11-17- Bovine) adenosine deaminase) Pegaspargase Oncaspar(monomethoxypolyethylene glycol succinimidyl L- asparaginase)Pegfilgrastim Neulasta (covalent conjugate of recombinant methionylhuman G- CSF (Filgrastim) and monomethoxypolyethylene glycol)Pentostatin Nipent Pipobroman Vercyte Plicamycin, Mithramycin Mithracin(antibiotic produced by Streptomyces plicatus) Porfimer sodium PhotofrinProcarbazine Matulane (N-isopropyl-μ-(2-methylhydrazino)-p-toluamidemonohydrochloride) Quinacrine Atabrine(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2- methoxyacridine)Rasburicase Elitek (recombinant peptide) Rituximab Rituxan (recombinantanti-CD20 antibody) Sargramostim Prokine (recombinant peptide)Streptozocin Zanosar (streptozocin 2-deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-a(and b)-D- glucopyranose and 220mg citric acid anhydrous) Talc Sclerosol (Mg₃Si₄O₁₀(OH)₂) TamoxifenNolvadex ((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8- carboxamide)teniposide, VM-26 Vumon (4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac(13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien-17-oic acid [dgr]-lactone)Thioguanine, 6-TG Thioguanine (2-amino-1,7-dihydro-6H-purine-6-thione)Thiotepa Thioplex (Aziridine, 1,1′,1″-phosphinothioylidynetris-, or Tris(1- aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7] indolizino [1,2-b] quinoline-3,14- (4H,12H)-dionemonohydrochloride) Toremifene Fareston(2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]-phenoxy)-N,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131 Tositumomab Bexxar(recombinant murine immunotherapeutic monoclonal IgG_(2a) lambdaanti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab Herceptin (recombinant monoclonal IgG₁ kappa anti-HER2antibody) Tretinoin, ATRA Vesanoid (all-trans retinoic acid) UracilMustard Uracil Mustard Capsules Valrubicin,N-trifluoroacetyladriamycin-14-valerate Valstar((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethylpentanoate) Vinblastine, Leurocristine Velban (C₄₆H₅₆N₄O₁₀•H₂SO₄)Vincristine Oncovin (C₄₆H₅₆N₄O₁₀•H₂SO₄) Vinorelbine Navelbine(3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine [R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronicacid Zometa ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acidmonohydrate)

Anticancer agents further include compounds which have been identifiedto have anticancer activity. Examples include, but are not limited to,3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007,ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100, alanosine,AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015,atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777,bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib,bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime,cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine,DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral,eflornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide,flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT,galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin,HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusionprotein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12,IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib,leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide,MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibodyJ591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin,nolatrexed dihydrochloride, nolvadex, NS-9, O6-benzylguanine, oblimersensodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin,PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone,PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase,rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4,rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992,SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilidehydroxamic acid, suramin, talabostat, talampanel, tariquidar,temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin,tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate,TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab,vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidartrihydrochloride.

For a more detailed description of anticancer agents and othertherapeutic agents, those skilled in the art are referred to any numberof instructive manuals including, but not limited to, the Physician'sDesk Reference and to Goodman and Gilman's “Pharmaceutical Basis ofTherapeutics” tenth edition, Eds. Hardman et al., 2002.

In some embodiments, methods provided herein, including the methodscomprising pulsatile dose administration, comprise administering one ormore compounds provided herein with radiation therapy. The methodsprovided herein are not limited by the types, amounts, or delivery andadministration systems used to deliver the therapeutic dose of radiationto an animal. For example, the animal may receive photon radiotherapy,particle beam radiation therapy, other types of radiotherapies, andcombinations thereof. In some embodiments, the radiation is delivered tothe animal using a linear accelerator. In still other embodiments, theradiation is delivered using a gamma knife.

The source of radiation can be external or internal to the animal.External radiation therapy is most common and involves directing a beamof high-energy radiation to a tumor site through the skin using, forinstance, a linear accelerator. While the beam of radiation is localizedto the tumor site, it is nearly impossible to avoid exposure of normal,healthy tissue. However, external radiation is usually well tolerated byanimals. Internal radiation therapy involves implanting aradiation-emitting source, such as beads, wires, pellets, capsules,particles, and the like, inside the body at or near the tumor siteincluding the use of delivery systems that specifically target cancercells (e.g., using particles attached to cancer cell binding ligands).Such implants can be removed following treatment, or left in the bodyinactive. Types of internal radiation therapy include, but are notlimited to, brachytherapy, interstitial irradiation, intracavityirradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g., metronidazole,misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR),nitroimidazole, 5-substituted-4-nitroimidazoles, 2H-isoindolediones,[[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol,nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins,halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazolederivatives, fluorine-containing nitroazole derivatives, benzamide,nicotinamide, acridine-intercalator, 5-thiotretrazole derivative,3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylatedtexaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine,carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine,etoposide, paclitaxel, heat (hyperthermia), and the like),radioprotectors (e.g., cysteamine, aminoalkyl dihydrogenphosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like).Radiosensitizers enhance the killing of tumor cells. Radioprotectorsprotect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to an animal, so long as thedose of radiation is tolerated by the animal without unacceptablenegative side-effects. Suitable types of radiotherapy include, forexample, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gammarays) or particle beam radiation therapy (e.g., high linear energyradiation). Ionizing radiation is defined as radiation comprisingparticles or photons that have sufficient energy to produce ionization,i.e., gain or loss of electrons (as described in, for example, U.S. Pat.No. 5,770,581 incorporated herein by reference in its entirety). Theeffects of radiation can be at least partially controlled by theclinician. In one embodiment, the dose of radiation is fractionated formaximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation administered to an animalis about 0.01 Gray (Gy) to about 100 Gy. In another embodiment, about 10Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy,45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course oftreatment. While in some embodiments a complete dose of radiation can beadministered over the course of one day, the total dose is ideallyfractionated and administered over several days. Desirably, radiotherapyis administered over the course of at least about 3 days, e.g., at least5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about1-8 weeks). Accordingly, a daily dose of radiation will compriseapproximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy,2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2Gy (e.g., 1.5-2 Gy). The daily dose of radiation should be sufficient toinduce destruction of the targeted cells. If stretched over a period, inone embodiment, radiation is not administered every day, therebyallowing the animal to rest and the effects of the therapy to berealized. For example, radiation desirably is administered on 5consecutive days, and not administered on 2 days, for each week oftreatment, thereby allowing 2 days of rest per week. However, radiationcan be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5days/week, 6 days/week, or all 7 days/week, depending on the animal'sresponsiveness and any potential side effects. Radiation therapy can beinitiated at any time in the therapeutic period. In one embodiment,radiation is initiated in week 1 or week 2, and is administered for theremaining duration of the therapeutic period. For example, radiation isadministered in weeks 1-6 or in weeks 2-6 of a therapeutic periodcomprising 6 weeks for treating, for instance, a solid tumor.Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of atherapeutic period comprising 5 weeks. These exemplary radiotherapyadministration schedules are not intended, however, to limit the methodsprovided herein.

Antimicrobial therapeutic agents may also be used as therapeutic agentsin combination with the compounds provided herein. Any agent that cankill, inhibit, or otherwise attenuate the function of microbialorganisms may be used, as well as any agent contemplated to have suchactivities. Antimicrobial agents include, but are not limited to,natural and synthetic antibiotics, antibodies, inhibitory proteins(e.g., defensins), antisense nucleic acids, membrane disruptive agentsand the like, used alone or in combination. Indeed, any type ofantibiotic may be used including, but not limited to, antibacterialagents, antiviral agents, antifungal agents, and the like.

In some embodiments of the methods provided herein, one or morecompounds provided herein and one or more therapeutic agents oranticancer agents are administered to an animal under one or more of thefollowing conditions: at different periodicities, at differentdurations, at different concentrations, by different administrationroutes, etc. In some embodiments, the compound is administered prior tothe therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12,or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior tothe administration of the therapeutic or anticancer agent. In someembodiments, the compound is administered after the therapeutic oranticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2,3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration ofthe anticancer agent. In some embodiments, the compound and thetherapeutic or anticancer agent are administered concurrently but ondifferent schedules, e.g., the compound is administered daily while thetherapeutic or anticancer agent is administered once a week, once everytwo weeks, once every three weeks, or once every four weeks. In otherembodiments, the compound is administered once a week while thetherapeutic or anticancer agent is administered daily, once a week, onceevery two weeks, once every three weeks, or once every four weeks.

In some embodiments, compositions provided herein comprise one or moreof the compounds provided herein in an amount which is effective toachieve its intended purpose. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typically, the compounds may be administered tomammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or anequivalent amount of the pharmaceutically acceptable salt thereof, perday of the body weight of the mammal being treated for disordersresponsive to induction of apoptosis. In one embodiment, about 0.01 toabout 25 mg/kg is orally administered to treat, ameliorate, or preventsuch disorders. For intramuscular injection, the dose is generally aboutone-half of the oral dose. For example, a suitable intramuscular dosewould be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5mg/kg.

The unit oral dose may comprise from about 0.01 to about 1000 mg, forexample, about 0.1 to about 100 mg of the compound. The unit dose may beadministered one or more times daily as one or more tablets or capsuleseach containing from about 0.1 to about 10 mg, conveniently about 0.25to 50 mg of the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a one embodiment, thecompound is present at a concentration of about 0.07-1.0 mg/ml, forexample, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, thecompounds provided herein may be administered as part of apharmaceutical preparation or composition. In some embodiments, thepharmaceutical preparation or composition can include one or morepharmaceutically acceptable carriers, excipients, and/or auxiliaries. Insome embodiments, the one or more carriers, excipients, and auxiliariesfacilitate processing of the compound into a preparation or compositionwhich can be used pharmaceutically. The preparations, particularly thosepreparations which can be administered orally or topically and which canbe used for one type of administration, such as tablets, dragees, slowrelease lozenges and capsules, mouth rinses and mouth washes, gels,liquid suspensions, hair rinses, hair gels, shampoos and alsopreparations which can be administered rectally, such as suppositories,as well as suitable solutions for administration by intravenousinfusion, injection, topically or orally, contain from about 0.01 to 99percent, in one embodiment from about 0.25 to 75 percent of activecompound(s), together with the one or more carriers, excipients, and/orauxiliaries.

The pharmaceutical compositions of provided herein may be administeredto any patient which may experience the beneficial effects of thecompounds provided herein. Foremost among such patients are mammals,e.g., humans, although the methods and compositions provided herein arenot intended to be so limited. Other patients include veterinary animals(cows, sheep, pigs, horses, dogs, cats and the like).

The compounds and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations provided herein are manufactured bymeans of conventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariescan be suitable flow-regulating agents and lubricants. Suitableauxiliaries include, for example, silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate, and/orpolyethylene glycol. Dragee cores are provided with suitable coatingswhich, if desired, are resistant to gastric juices. For this purpose,concentrated saccharide solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycoland/or titanium dioxide, lacquer solutions and suitable organic solventsor solvent mixtures. In order to produce coatings resistant to gastricjuices, solutions of suitable cellulose preparations such asacetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate,are used. Dye stuffs or pigments may be added to the tablets or drageecoatings, for example, for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are in oneembodiment dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions provided herein are formulated in oneembodiment as oils, creams, lotions, ointments and the like by choice ofappropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The carriers may be those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight. Lotions may be conveniently prepared by dissolvingthe active ingredient, in a suitable high molecular weight alcohol suchas propylene glycol or polyethylene glycol.

In one embodiment, the present disclosure relates to methods of treatinga patient with a hyperproliferative disease, e.g., cancer, the methodscomprising pulsatile administration to the patient one or more compoundsor compositions provided herein, or pharmaceutically acceptable salts,solvates, or prodrugs thereof.

The following examples are illustrative, but not limiting, of thecompounds, compositions, and methods provided herein. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art are within the spirit and scope ofthe methods, compounds, and compositions provided herein.

In certain aspects, the following embodiments are provided:

Embodiment I

A method of treating, preventing, or ameliorating cancer in a patient,wherein the method comprises pulsatile administration to the patient atherapeutically effective amount of a compound having Formula XII:

or a pharmaceutically acceptable salt thereof,wherein:R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:R^(6a), R^(6b), R^(6c), R^(6d), and R^(6e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;R³ is optionally substituted C₁-C₈alkyl;R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;R⁵ is selected from the group consisting of:

wherein:R⁷ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;X is selected from the group consisting of O, S, and NR′;Y is selected from the group consisting of O, S, and NR″;R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; andR″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl,wherein the compound is substantially free of one or more otherstereoisomers.

Embodiment II

The method of Embodiment I, wherein R⁴ is hydrogen, or apharmaceutically acceptable salt thereof.

Embodiment III

The method of Embodiment I, wherein X is NH, or a pharmaceuticallyacceptable salt thereof.

Embodiment IV

The method of Embodiment I, wherein Y is NH, or a pharmaceuticallyacceptable salt thereof.

Embodiment V

The method of Embodiment I, wherein R³ is —CH₂C(CH₃)₃, or apharmaceutically acceptable salt thereof.

Embodiment VI

The method of Embodiment I, wherein R⁵ is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.

Embodiment VII

The method of Embodiment I, wherein:

R^(1a) is hydrogen;R^(1b), R^(1c), and R^(1d) are each independently selected from thegroup consisting of hydrogen, fluoro, and chloro;R³ is C₄-C₈ alkyl;R⁴ is hydrogen;R⁵ is selected from the group consisting of:

X and Y are NH;

or a pharmaceutically acceptable salt thereof.

Embodiment VIII

The method of Embodiments VI or VII, wherein R⁵ is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

Embodiment IX

The method of Embodiment I, wherein the compound of Formula XII isselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Embodiment X

The method of Embodiment I, wherein the compound of Formula XII is:

or a pharmaceutically acceptable salt thereof.

Embodiment XI

The method of Embodiment I, wherein the compound of Formula XII is:

or a pharmaceutically acceptable salt thereof.

Embodiment XII

The method of any one of Embodiments IX-XI, wherein the compound issubstantially free of other stereoisomers, or a pharmaceuticallyacceptable salt thereof.

Embodiment XIII

The method of Embodiment XII, wherein the compound is a substantiallypure stereoisomer, or a pharmaceutically acceptable salt thereof.

Embodiment XIV

The method of any one of Embodiments I-XIII, wherein the compound isadministered to the patient one day a week, one day every two weeks, oneday every three weeks, or one day every four weeks.

Embodiment XV

The method of any one of Embodiments I-XIV, wherein cells of thehyperproliferative disease express functional p53.

Embodiment XVI

The method of any one of Embodiments I-XV, wherein thehyperproliferative disease is cancer.

Embodiment XVII

The method of Embodiment XVI, further comprising administering to thepatient one or more anticancer agents.

Embodiment XVIII

The method of Embodiment XVII, wherein the anticancer agent is achemotherapeutic agent.

Embodiment XIX

The method of Embodiment XVIII, wherein the anticancer agent isradiation therapy.

Embodiment XX

A kit comprising a compound having Formula XII:

or a pharmaceutically acceptable salt thereof,wherein:R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:R^(6a), R^(6b), R^(6c), R^(6d), and R^(6e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;R³ is optionally substituted C₁-C₈ alkyl;R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;R⁵ is selected from the group consisting of:

wherein:R⁷ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;X is selected from the group consisting of O, S, and NR′;Y is selected from the group consisting of O, S, and NR″;R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; andR″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl,wherein the compound is substantially free of one or more otherstereoisomers,and instructions for pulsatile administration of the compound to apatient having a hyperproliferative disease.

Embodiment XXI

The kit of Embodiment XX, wherein the hyperproliferative disease iscancer.

Embodiment XXII

The kit of Embodiment XXI, further comprising one or more anticanceragents.

Embodiment XXIII

The kit of Embodiment XXII, wherein the instructions directco-administration of the compound together with the one or moreanticancer agents.

In certain aspects, the following particular embodiments are providedherein:

Embodiment XXIV

A method of treating, ameliorating, or preventing melanoma in a patientcomprising administering to the patient a therapeutically effectiveamount of a compound having Formula XII:

wherein:R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:R^(6a), R^(6b), R^(6c), R^(6d), and R^(6e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;R³ is optionally substituted C₁-C₈ alkyl;R⁴ is selected from the group consisting of hydrogen andoptionally-substituted C₁-C₆ alkyl;R⁵ is selected from the group consisting of:

wherein:R⁷ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;X is selected from the group consisting of O, S, and NR′;Y is selected from the group consisting of O, S, and NR″;R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; andR″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl,wherein the compound is substantially free of one or more otherstereoisomers,or a pharmaceutically acceptable salt thereof.

Embodiment XXV

The method of Embodiment XXIV, further comprising administering to thepatient one or more additional anticancer agents.

Embodiment XXVI

The method of Embodiment XXV, wherein the anticancer agent is achemotherapeutic agent.

Embodiment XXVII

The method of Embodiment XXVI, wherein the anticancer agent is radiationtherapy.

Embodiment XXVIII

The method of any one of Embodiments XXIV-XXVII, wherein the melanoma ischaracterized by resistance to conventional cancer therapy.

Embodiment XXIX

The method of any one of Embodiments XXIV-XXVIII, wherein the melanomaexpresses wild-type p53 protein.

Embodiment XXX

The method of any one of Embodiments XXIV-XXIX, wherein the compound ofFormula XII is:

or a pharmaceutically acceptable salt thereof.

Embodiment XXXI

A kit comprising a compound having Formula XII:

wherein:R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:R^(6a), R^(6b), R^(6c), R^(6d), and R^(6e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;R³ is optionally substituted C₁-C₈ alkyl;R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;R⁵ is selected from the group consisting of:

wherein:R⁷ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;X is selected from the group consisting of O, S, and NR′;Y is selected from the group consisting of O, S, and NR″;R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; andR″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl,wherein the compound is substantially free of one or more otherstereoisomers,or a pharmaceutically acceptable salt thereof,and instructions for administering the compound to a patient havingmelanoma.

Embodiment XXXII

The kit of Embodiment XXXI, further comprising one or more additionalanticancer agents.

Embodiment XXXIII

The kit of Embodiment XXXI, wherein the instructions directco-administration of the compound together with an additional anticanceragent.

In certain aspects, the following particular embodiments are providedherein:

Embodiment XXXIV

A compound having Formulae XII:

wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

R² is:

wherein:

R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro;

R³ is optionally substituted C₁-C₈ alkyl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl; and

R″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl,

wherein the compound is substantially free of one or more otherstereoisomers,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XXXV

The compound of Embodiment XXXIV, wherein R⁴ is hydrogen, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XXXVI

The compound of Embodiment XXXIV, wherein X is NH, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

Embodiment XXXVII

The compound of Embodiment XXXIV, wherein Y is NH, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

Embodiment XXXVIII

The compound of Embodiment XXXIV, wherein R³ is —CH₂C(CH₃)₃, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XXXIX

The compound of Embodiment XXXIV, wherein R⁵ is selected from the groupconsisting of:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XL

The compound of Embodiment XXXVIII, wherein:

R^(1a) is hydrogen;R^(1b), R^(1c), and R^(1d) are each independently selected from thegroup consisting of hydrogen, fluoro, and chloro;R³ is C₄-C₈ alkyl;R⁴ is hydrogen;R⁵ is selected from the group consisting of:

and

X and Y are NH;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XLI

The compound of Embodiments XXXIX or XL, wherein R⁵ is selected from thegroup consisting of:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XLII

The compound of Embodiment XXXIV selected from the group consisting of:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XLIII

A compound having the structure:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XLIV

A compound having the structure:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Embodiment XLV

The compound of any one of Embodiments XLII-XLIV, wherein the compoundis substantially free of other stereoisomers, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

Embodiment XLVI

The compound of Embodiment XLV, wherein the compound is a substantiallypure stereoisomer, or a pharmaceutically acceptable salt, solvate, orprodrug thereof.

Embodiment XLVII

A pharmaceutical composition comprising the compound of any one ofEmbodiments XXXIV-XLVI and a pharmaceutically acceptable carrier.

Embodiment XLVIII

A method of treating a patient comprising administering to the patient atherapeutically effective amount of the compound or a pharmaceuticallyacceptable salt thereof of any one of Embodiments XXXIV-XLVI, whereinthe patient has a hyperproliferative disease.

Embodiment XLIX

A method of treating a patient comprising administering to the patient atherapeutically effective amount of the pharmaceutical composition ofEmbodiment XLVII, wherein the patient has a hyperproliferative disease.

Embodiment L

The method of Embodiments XLVIII or XLIX, wherein the hyperproliferativedisease is cancer.

Embodiment LI

The method of claims Embodiments XLVIII or XLIX, wherein cells of thehyperproliferative disease express functional p53.

Embodiment LII

The method of Embodiment L, further comprising administering to thepatient one or more anticancer agents.

Embodiment LIII

The method of Embodiment LII, wherein the anticancer agent is achemotherapeutic agent.

Embodiment LIV

The method of Embodiment LII, wherein the anticancer agent is radiationtherapy.

Embodiment LV

A method of treating a patient, wherein the patient has ahyperproliferative disorder and is being treated with an anticanceragent, comprising administering to the patient a compound orpharmaceutically acceptable salt thereof of any one of EmbodimentsXXXIV-XLVI.

Embodiment LVI

The method of Embodiment LV, wherein the patient is experiencingside-effects of the anticancer agent treatment selected from the groupconsisting of mucositis, stomatitis, xerostoma, alopecia, andgastrointestinal disorder.

Embodiment LVII

The method of Embodiment LVI, wherein cells of the hyperproliferativedisorder express functional p53.

Embodiment LVIII

A kit comprising a compound of any one of Embodiments XXXIV-XLVI andinstructions for administering the compound to a patient having ahyperproliferative disease.

Embodiment LIX

The kit of Embodiment LVIII, wherein the hyperproliferative disease iscancer.

Embodiment LX

The kit of Embodiment LIX, further comprising one or more anticanceragents.

Embodiment LXI

The kit of Embodiment LX, wherein the instructions directco-administration of the compound together with the one or moreanticancer agents.

Example 1 Analytical Data for Compounds General Information

NMR spectra were recorded on a BRUKER AVANCE 250, BRUKER AVANCE 300,BRUKER AVANCE DRX-400, or BRUKER AVANCE DPX-500, or similar instrument.Unless otherwise indicated all NMR chemical shifts reported herein aredenoted by the delta (δ) scale.

Liquid chromatography-mass spectrometry (denoted “LC-MS”) analysis wasperformed using method A, method B, or method C:

Method A: WATERS HPLC-SQD apparatus; Ionization: electrospray inpositive mode and/or negative mode (ES+/−); Chromatographic conditions:Column: ACQUITY BEH C18 1.7 μm-2.1×50 mm; Solvents: A: H₂O (0.1% formicacid) B: CH₃CN (0.1% formic acid); Column temperature: 50° C.; Flow: 1ml/min; Gradient (2 min): from 5 to 50% of B in 0.8 min; 1.2 min: 100%of B; 1.85 min: 100% of B; 1.95: 5% of B; Retention time=t_(R) (min).

Method B: WATERS ZQ apparatus; Ionization: electrospray in positive modeand/or negative mode (ES+/−); Chromatographic conditions: Column:XBridge C18 2.5 μm—3×50 mm; Solvents: A: H₂O (0.1% formic acid) B: CH₃CN(0.1% formic acid); Column temperature: 70° C.; Flow: 0.9 ml/min;Gradient (7 min): from 5 to 100% of B in 5.3 min; 5.5 min: 100% of B;6.3 min: 5% of B; Retention time=t_(R) (min).

Method C: WATERS UPLC-SQD apparatus; Ionization: electrospray inpositive mode and/or negative mode (ES+/−); Chromatographic conditions:Column: ACQUITY BEH C18 1.7 μm—2.1×50 mm; Solvents: A: H₂O (0.1% formicacid) B: CH₃CN (0.1% formic acid); Column temperature: 50° C.; Flow: 0.8ml/min; Gradient (2.5 min): from to 100% of B in 1.8 min; 2.4 min: 100%of B; 2.45 min: 100% of B; from 100 to 5% of B in 0.05 min; Retentiontime=t_(R) (min).

Purity analysis was performed using reverse-phase HPLC using a SunFire™C18 5 μm 4.6×150 mm column at a flow rate of 1 mL/min under thefollowing conditions: Condition I: Gradient from 90% of solvent A (0.1%of TFA in water) and 10% of solvent B (0.1% of TFA in methanol) to 5% ofsolvent A and 95% of solvent B in 85 min; and Condition II: Gradientfrom 80% of solvent A (0.1% of TFA in water) and 20% of solvent B (0.1%of TFA in acetonitrile) to 50% of solvent A and 50% of solvent B in 30min.

Low resolution ESI mass spectrum analysis was performed onThermo-Scientific LCQ Fleet mass spectrometer or similar instrument.

The chemical names of the compounds provided in this example weredetermined with ADCLABS version 12.0.

¹H NMR (300 MHz, MeOH-d₄): 7.50-7.36 (m, 1H), 7.24-7.10 (m, 2H),6.88-6.76 (m, 3H), 5.12 (d, J=10.17 Hz, 1H), 4.49 (d, J=10.17 Hz, 1H),4.23 (dd, J=6.83, 2.09 Hz, 1H), 3.98-3.83 (m, 1H), 2.49-2.36 (m, 1H),2.36-2.22 (m, 1H), 2.10-1.96 (m, 2H), 1.94-1.82 (m, 1H), 1.35-1.28 (m,1H), 1.29 (s, 3H), 0.80 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 108.1,166.0, 145.4, 136.9, 127.9, 126.1 (t, J_(C-F)=5.6 Hz), 125.4, 123.4118.8 (d, J_(C-F)=17.3 Hz), 112.0, 67.4, 64.5, 63.7, 61.6, 49.5, 45.6,45.5, 42.4, 38.5, 30.9, 29.5, 27.6; ESI-MS calculated for C₂₈H₃₃³⁵ClF₂N₃O₃ [M+H]⁺: 532.2179. Found: 532.42.

C029—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.84 (d, J=6.80 Hz, 1H), 7.58 (t, J=6.80 Hz,1H), 7.39 (t, J=7.11 Hz, 1H), 7.22 (t, J=7.80 Hz, 1H), 6.88 (dd, J=9.81,7.80 Hz, 1H), 6.78 (d, J=10.13, 6.63 Hz, 1H), 5.11 (d, J=10.37 Hz, 1H),4.48 (d, J=10.37 Hz, 1H), 4.21 (d, J=10.37 Hz, 1H), 4.21 (dd, J=7.32,2.66 Hz, 1H), 3.95-3.75 (m, 1H), 2.46-2.22 (m, 2H), 2.12-1.96 (m, 2H),1.94-1.80 (m, 1H), 1.34-1.28 (m, 1), 1.29 (s, 3H), 0.81 (s, 9H); ¹³C NMR(75 MHz, MeOH-d₄): 180.2, 169.2, 132.2, 128.7 (d, J_(C-F)=2.2 Hz), 126.5(d, J_(C-F)=4.6 Hz), 124.7 (dd, J_(C-F)=33.5, 19.2 Hz), 122.6 (d,J_(C-F)=18.1 Hz), 101.5 (d, J_(C-F)=23.0 Hz), 67.4, 64.4, 63.5, 61.9,49.8, 45.6, 45.5, 42.4, 38.6, 30.9, 29.5, 27.6; ESI-MS calculated forC₂₈H₃₂ ³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.33.

C031—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.68-7.54 (m, 1H), 7.38-7.26 (m, 1H),7.22-7.12 (m, 1H), 6.90-6.76 (m, 1H), 6.70-6.60 (m, 1H), 6.56-6.42 (m,1H), 5.30-5.20 (m, 1H), 4.49 (d, J=10.03 Hz, 1H), 4.25 (dd, J=71.9, 2.39Hz, 1H), 4.00-3.82 (m, 1H), 2.50-2.21 (m, 2H), 2.18-2.00 (m, 2H),1.98-1.82 (m, 1H), 1.40-1.30 (m, 1H), 1.28 (s, 3H), 0.79 (s, 9H); ¹³CNMR (75 MHz, MeOH-d₄): 180.6, 165.1 (d, J_(C-F)=246.7 Hz), 166.1, 157.7(d, J_(C-F)=247.9 Hz), 145.6 (d, J_(C-F)=12.0 Hz), 132.0, 128.6, 128.2(d, J_(C-F)=10.2 Hz), 126.3 (d, J_(C-F)=4.5 Hz), 125.0 (d, J_(C-F)=14.0Hz), 122.4 (d, J_(C-F)=18.4 Hz), 122.3, 109.8 (d, J_(C-F)=23.2 Hz), 99.9(d, J_(C-F)=27.8 Hz), 67.4, 64.5, 63.5, 61.5, 49.2, 45.6, 45.5, 42.3,38.4, 30.9, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃ ³⁵ClF₂N₃O₃ [M+H]⁺:532.2179. Found: 532.42.

C034—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.28-7.10 (m, 5H), 6.92-6.84 (m, 1H),6.80-6.76 (m, 1H), 5.40-5.20 (m, 1H), 5.08 (d, J=10.96 Hz, 1H),4.40-4.20 (m, 1H), 3.90-3.60 (m, 1H), 2.50-2.30 (m, 1H), 2.30-2.15 (m,1H), 2.15-2.00 (m, 2H), 1.90-1.75 (m, 1H), 1.57 (dd, J=15.3, 3.71 Hz,1H), 1.25 (s, 3H), 0.79 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 180.0,165.9, 144.7, 136.7, 136.6, 135.8, 131.3, 130.1, 129.8, 128.1, 128.1,126.8, 123.5, 112.0, 67.4, 64.3, 64.0, 62.2, 57.2, 45.7, 45.6, 42.7,38.3, 31.0, 29.6, 27.5; ESI-MS calculated for C₂₈H₃₄ ³⁵Cl₂N₃O₃ [M+H]⁺:530.1977. Found: 530.50.

C035—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.40-7.00 (m, 5H), 6.80-6.40 (m, 1H),5.60-5.00 (m, 2H), 4.60-4.20 (m, 1H), 4.00-3.80 (m, 1H), 2.60-2.40 (m,1H), 2.40-2.20 (m, 1H), 2.20-2.00 (m, 2H), 2.00-1.80 (m, 1H), 1.70-1.50(m, 1H), 1.28 (s, 3H), 0.83 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 180.0,165.8, 160.0-145.0 (m, 2×C_(sp2)-F), 136.5, 135.9, 131.4, 130.0, 129.9,128.0, 124.1 (d, J_(C-F)=6.3 Hz), 119.1, 116.7 (d, J_(C-F)=20.4 Hz),101.4 (d, J_(C-F)=23.0 Hz), 67.4, 64.2, 63.8, 62.5, 57.4, 45.6, 45.5,42.7, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃ ³⁵ClF₂N₃O₃[M+H]⁺: 532.2179. Found: 532.42.

MI-519-73—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.50-7.30 (m, 2H), 7.20-7.10 (m, 1H),6.90-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.36 (d, J=9.76 Hz, 1H), 4.05-3.96(m, 1H), 3.70-3.50 (m, 1H), 1.94 (dd, J=14.98, 7.30 Hz, 1H), 1.80-1.00(m, 8H), 1.16 (s, 3H), 0.90-0.70 (m, 1H), 0.80 (s, 9H); ESI-MScalculated for C₃₀H₃₇ ³⁵Cl₂FN₃O₃ [M+H]⁺: 576.2196. Found: 576.58.

MI-519-74—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.50-7.30 (m, 2H), 7.25-7.10 (m, 1H),6.85-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.32 (d, J=9.69 Hz, 1H), 4.10-3.95(m, 1H), 3.85-3.70 (m, 1H), 2.00-1.80 (m, 2H), 1.75-1.20 (m, 7H), 1.13(s, 3H), 0.95-0.75 (m, 1H), 0.81 (s, 9H); ESI-MS calculated for C₃₀H₃₇³⁵Cl₂FN₃O₃ [M+H]⁺: 576.2196. Found: 576.58.

MI-7102—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.36-7.25 (m, 1H), 7.24-7.11 (m, 2H), 6.86(d, J=1.8 Hz, 1H), 6.80 (dd, J=1.8, 8.1 Hz, 1H), 6.72 (d, J=8.1 Hz, 1H),4.82 (d, J=9.6 Hz, 1H), 4.36 (d, J=9.6 Hz, 1H), 4.04 (dd, J=2.4, 7.4 Hz,1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.05-1.78 (m, 5H), 1.75-1.59(m, 1H), 1.43-1.04 (m, 5H), 0.81 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58; HPLC (Condition I)t_(R)=50.45 min (Purity 95.4%).

MI-7103—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.38 (d, J=7.7 Hz, 1H), 7.54 (t, J=6.7 Hz,1H), 7.40 (d, J=7.1 Hz, 1H), 7.20 (t, J=7.9 Hz, 1H), 6.93 (d, J=6.1 Hz,1H), 6.86 (d, J=8.7 Hz, 1H), 4.45 (d, J=10.3 Hz, 1H), 4.13 (dd, J=2.8,7.5 Hz, 1H), 3.77-3.55 (m, 1H), 3.55-3.42 (m, 1H), 2.09-1.71 (m, 4H),1.70-1.56 (m, 1H), 1.4₅-1_(.0)2 (m, 5 _(H)), 0.82 (s, 9H); ESI-MScalculated for C₂₉H₃₄Cl₂F2N₃O₃ (M+H)⁺ requires 580.19. found 580.67;HPLC (Condition I) t_(R)=55.01 min (Purity 88.1%).

MI-7104—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.49 (t, J=7.2 Hz, 1H), 7.45-7.38 (m, 1H),7.22 (t, J=8.0 Hz, 1H), 6.85-6.68 (m, 2H), 4.80 (d, J=9.8 Hz, 1H), 4.36(d, J=9.9 Hz, 1H), 4.01 (dd, J=2.4, 7.6 Hz, 1H), 3.74-3.57 (m, 1H),3.55-3.39 (m, 1H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1H), 1.44-1.04 (m,5H), 0.90 (d, J=4.5 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated forC₂₉H₃₄ClF₃N₃O₃ (M+H)⁺ requires 564.22. found 564.58; HPLC (Condition I)t_(R)=51.76 min (Purity 86.9%).

MI-7105—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.49 (t, J=7.2 Hz, 1H), 7.45-7.38 (m, 1H),7.22 (t, J=8.0 Hz, 1H), 6.85-6.68 (m, 2H), 4.80 (d, J=9.8 Hz, 1H), 4.36(d, J=9.9 Hz, 1H), 4.01 (dd, J=2.4, 7.6 Hz, 1H), 3.74-3.57 (m, 1H),3.55-3.39 (m, 1H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1H), 1.44-1.04 (m,5H), 0.90 (d, J=4.5 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58; HPLC (Condition I)t_(R)=49.20 min (Purity 99.4%).

MI-7106—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.36 (d, J=7.0 Hz, 1H), 7.59 (d, J=8.1 Hz,1H), 7.41-7.11 (m, 4H), 7.04 (d, J=7.6 Hz, 1H), 6.78 (d, J=1.8 Hz, 1H),5.19 (d, J=11.3 Hz, 1H), 4.44 (J=8.1 Hz, 1H), 4.07 (d, J=11.3 Hz, 1H),3.74-3.53 (m, 1H), 3.53-3.37 (m, 1H), 2.08-_(1.)8₃ (m, 3_(H)), 1.83-1.⁶9(m, 1H), 1.61-1.44 (m, 1H), 1.44-1.08 (m, 4H), 1.07-0.72 (m, 1H), 0.88(s, 9H); ESI-MS calculated for C₂₉H₃₆Cl₂N₃O₃ (M+H)⁺ requires 544.21.found 544.67; HPLC (Condition I) t_(R)=51.41 min (Purity 93.0%).

MI-7108—TFA Salt

¹H NMR (300 MHz, MeOH-d₄/DMSO-d₆): 10.15 (s, 1H), 7.76 (d, J=8.2 Hz,1H), 7.22 (s, 1H), 7.17-7.00 (m, 3H), 6.94 (d, J=7.1 Hz, 1H), 6.81 (d,J=6.0 Hz, 1H), 4.42 (d, J=8.3 Hz, 1H), 4.09 (d, J=3.0 Hz, 1H), 3.79 (d,J=8.3 Hz, 1H), 3.73-3.49 (m, 2H), 3.35 (d, J=9.5 Hz, 1H), 2.10-1.84 (m,4H), 1.52-1.11 (m, 5H), 0.87 (s, 9H); 13C NMR (75 MHz, MeOH-d₄/DMSO-d₆):177.1, 172.4, 153.6 (d, J_(C-F)=242.7 Hz), 138.7, 138.5 (d, J_(C-F)=2.4Hz), 133.2, 129.0, 127.544, 127.541 (d, J_(C-F)=6.7 Hz), 126.8, 126.5,119.7 (d, J_(C-F)=19.2 Hz), 111.3, 110.4 (d, J_(C-F)=24.1 Hz), 68.4,66.6, 65.7, 64.0, 58.6, 46.8, 42.2, 33.26, 33.20, 30.4, 30.2, 29.7,29.5; ESI-MS calculated for C₂₉H₃₅Cl₂FN₃O₃ (M+H)⁺ requires 562.20. found562.67; HPLC (Condition I) t_(R)=55.08 min (Purity 96.1%); HPLC(Condition II) t_(R)=21.44 min (Purity 92.7%).

MI-7109—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.47 (t, J=6.7 Hz, 1H), 7.42-7.33 (m, 1H),7.18 (t, J=7.7 Hz, 1H), 6.87 (d, J=1.8 Hz, 1H), 6.78 (dd, J=1.8, 8.1 Hz,1H), 6.70 (d, J=8.1 Hz, 1H), 4.40 (d, J=9.7 Hz, 1H), 4.11 (dd, J=2.5,7.6 Hz, 1H), 2.77-2.65 (m, 1H), 1.99 (dd, J=7.6, 15.3 Hz, 1H), 1.24 (dd,J=2.5, 15.3 Hz, 1H), 0.92-0.62 (m, 2H), 0.81 (s, 9H), 0.56-0.30 (m, 2H);ESI-MS calculated for C₂₆H₂₉Cl₂FN₃O₂ (M+H)⁺ requires 504.1₆. found504.58; HPLC (Condition I) t_(R)=53.99 min (Purity 94.4%).

B059—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.45-7.34 (m, 1H), 7.26-7.12 (m, 1H), 7.04-6.93(m, 1H), 6.90 (d, J=1.80 Hz, 1H), 6.65 (dd, J=8.08, 1.80 Hz, 1H), 4.41(d, J=9.25 Hz, 1H), 3.96 (quint, J=8.13 Hz, 1H), 2.51-2.07 (m, 2H),2.40-2.20 (m, 2H), 1.88 (dd, J=14.20, 9.91 Hz, 1H), 1.32 (s, 3H),1.20-0.80 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, CD₃OD): 181.3, 172.9(d, J_(C-F)=266.9 Hz), 168.6, 162.7, 145.3, 135.8, 131.7, 130.7 (d,J_(C-F)=38.6 Hz), 126.2 (d, J_(C-F)=4.5 Hz), 126.1, 123.6, 122.9, 122.7,111.4, 78.4, 67.7, 63.4, 46.0, 45.8, 44.3, 38.0, 31.4, 30.2, 27.6;ESI-MS calculated for C₂₈H₃₁₃₅Cl₂FN₃O₃ [M+H]⁺: 546.1727. Found: 546.50.

MI-519-77—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.50-7.40 (m, 1H), 7.40 (m, 1H), 7.20-7.10 (m,1H), 6.85 (d, J=1.40 Hz, 1H), 6.84-6.72 (m, 2H), 5.00-4.80 (m, 1H), 4.45(d, J=10.10 Hz, 1H), 4.02 (t, J=6.61 Hz, 1H), 3.90 (quintet, J=8.07 Hz,1H), 2.50-2.25 (m, 2H), 2.10-1.82 (m, 3H), 1.81-1.31 (m, 8H), 1.30 (s,3H), 1.10-0.91 (m, 1H), 0.91-0.81 (m 1H); ESI-MS calculated for C₂₉H₃₃³⁵Cl₂FN₃O₃ [M+H]⁺: 560.1883. Found: 560.50.

MI-519-78—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.45-7.31 (m, 2H), 7.20-7.11 (m, 1H), 6.86-6.82(m, 1H), 6.81-6.78 (m, 2H), 4.90-4.80 (m, 1H), 4.45 (d, J=10.33 Hz, 1H),4.10-3.95 (m, 1H), 3.70-3.60 (m, 1H), 3.50-3.40 (m, 1H), 2.10-1.05 (m,17H), 1.05-0.95 (m, 1H), 0.95-0.80 (m, 1H); ESI-MS calculated for C₃₀H₃₅³⁵Cl₂FN₃O₃ [M+H]⁺: 574.2040. Found: 574.58.

MI-519-80—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.80-7.72 (m, 1H), 7.50-7.38 (m, 2H), 6.87 (d,J=1.81 Hz, 1H), 6.71 (dd, J=8.16, 1.81 Hz, 1H), 6.52-6.40 (m, 1H),4.96-4.80 (m, 1H), 4.62 (d, J=8.69 Hz, 1H), 4.10-3.95 (m, 1H), 3.70-3.55(m, 1H), 3.50-3.45 (m, 1H), 2.00-1.80 (m, 3H), 1.80-1.60 (m, 1H),1.40-1.00 (m, 5H), 0.95-0.85 (m, 1H), 0.80 (s, 9H); ESI-MS calculatedfor C₂₉H₃₅ ³⁵Cl₃N₃O₃ [M+H]⁺: 578.1744. Found: 578.75.

C02701—TFA Salt(2′S,3′R,4′S,5′R)-6-Chloro-4′-(2,3-difluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 8.82 (d, J=6.83 Hz, 1H), 7.65-7.55 (m, 1H),7.45-7.30 (m, 1H), 7.20-7.05 (m, 3H), 6.80-6.75 (m, 1H), 5.40-5.10 (m,1H), 4.61 (d, J=11.39 Hz, 1H), 4.50 (d, J=7.66 Hz, 1H), 3.95-3.80 (m,1H), 2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.80 (m, 2H), 1.80-1.60(m, 1H), 1.27 (s, 3H), 1.20-1.08 (m, 1H), 0.86 (s, 9H); ¹³C NMR (75 MHz,MeOH-d₄): 177.8, 167.0, 160.0-148.0 (m, 2×C_(sp2)-F), 145.2, 137.2,126.8, 126.5-126.0 (m), 125.0, 124.1, 123.5, 122.1 (d, J_(C-F)=9.74 Hz),119.1 (d, J_(C-F)=17.1 Hz), 112.1, 67.3, 64.5, 64.2, 62.6, 48.5, 45.6,45.5, 43.3, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃³⁵ClF₂N₃O₃ [M+H]⁺: 532.2179. Found: 532.50.

LC-MS: t_(R) (min)=0.86; [M+H]⁺: m/z 532; [M−H]⁻: m/z 530 (method A).

¹H NMR (400 MHz, DMSO-d₆+TFA): 0.80 (s, 9H); 1.01 (d broad, J=15.2 Hz, 1H); 1.20 (s, 3H); 1.60 (m, 1H); 1.85 to 1.98 (m, 2H); 2.08 (m, 1H); 2.25(m, 1H); 3.72 (m, 1H); 4.49 (m, 2H); 5.30 (d, J=12.1 Hz, 1H); 6.78 (d,J=2.0 Hz, 1H); 7.16 (dd, J=2.0 and 8.3 Hz, 1H); 7.23 (m, 1H); 7.42 (m,1H); 7.48 (m, 1H); 7.74 (d, J=8.3 Hz, 1H).

C02901—TFA Salt(2′S,3′R,4′S,5′R)-4′-(3-Chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-5,6-difluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.80-7.65 (m, 1H), 7.60-7.50 (m, 1H),7.40-7.30 (m, 1H), 7.20-7.10 (m, 1H), 6.80-6.65 (m, 1H), 5.50-5.10 (m,1H), 4.60 (d, J=11.39 Hz, 1H), 4.50 (d, J=6.96 Hz, 1H), 3.95-3.80 (m,1H), 2.50-2.30 (m, 1H), 2.30-2.20 (m, 1H), 2.10-1.80 (m, 2H), 1.80-1.65(m, 1H), 1.27 (s, 3H), 1.20-1.05 (m, 1H), 0.87 (s, 9H); ¹³C NMR (75 MHz,MeOH-d₄): 177.8, 167.0, 160.0-145.0 (m, 3×C_(sp2)-F), 132.6, 128.6,126.6, 122.5 (d, J_(C-F)=18.9 Hz), 121.3 (d, J_(C-F)=13.0 Hz), 118.8,115.4 (d, J_(C-F)=21.7 Hz), 115.1, 101.8 (d, J_(C-F)=23.3 Hz), 67.3,64.6, 64.3, 62.5, 48.7, 45.6, 45.5, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MScalculated for C₂₈H₃₂ ³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.33.

LC-MS: t_(R) (min)=0.87; [M+H]⁺: m/z 550; [M−H]⁻: m/z 548 (method A).

¹H NMR (400 MHz; DMSO-d₆+TFA): 0.80 (s, 9H); 1.01 (d, J=15.2 Hz, 1H);1.20 (s, 3H); 1.62 (m, 1H); 1.85 to 1.98 (m, 2H); 2.08 (m, 1H); 2.26 (m,1H); 3.73 (m, 1H); 4.52 (m, 2H); 5.28 (d, J=12.1 Hz, 1H); 6.79 (dd,J=6.7 and 10.1 Hz, 1H); 7.25 (t, J=7.8 Hz, 1H); 7.50 (m, 1H); 7.60 (m,1H); 8.08 (m, 1H).

C03001—TFA Salt(2′S,3′R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-5-fluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.70 (d, J=7.30 Hz, 1H), 7.60-7.50 (m, 1H),7.45-7.35 (m, 1H), 7.25-7.15 (m, 1H), 6.88 (d, J=6.00 Hz, 1H), 5.21 (d,J=11.35 Hz, 1H), 4.61 (d, J=11.37 Hz, 1H), 4.53 (d, J=8.19 Hz, 1H),3.95-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.15 (m, 1H), 2.00-1.80 (m,2H), 1.80-1.60 (m, 1H), 1.29 (s, 3H), 1.25-1.05 (m, 1H), 0.89 (s, 9H);¹³C NMR (75 MHz, MeOH-d₄): 177.3, 166.7, 157.6 (d, J_(C-F)=249.5 Hz),155.7 (d, J_(C-F)=243.5 Hz), 140.4 (d, J_(C-F)=2.8 Hz), 132.5, 128.4,126.4 (d, J_(C-F)=4.9 Hz), 125.0 (d, J_(C-F)=7.4 Hz), 123.4 (d,J_(C-F)=19.5 Hz), 122.3 (d, J_(C-F)=18.9 Hz), 121.0 (d, J_(C-F)=13.0Hz), 114.5 (d, J_(C-F)=25.1 Hz), 104.8, 67.1, 64.6, 64.2, 62.4, 47.3,45.4, 45.3, 43.2, 38.2, 30.8, 29.2, 27.3; ESI-MS calculated forC₂₈H₃₂₃₅Cl₂F₂N₃O₃ [M+H]⁺: 566.1789. Found: 566.50.

LC-MS: t_(R) (min)=0.93; [M+H]⁺: m/z 566 (method A).

¹H NMR (400 MHz, DMSO-d₆+TFA): 0.81 (s, 9H); 1.02 (d broad, J=15.2 Hz, 1H); 1.20 (s, 3H); 1.62 (m, 1H); 1.87 to 1.99 (m, 2H); 2.09 (m, 1H); 2.27(m, 1H); 3.75 (m, 1H); 4.55 (m, 2H); 5.30 (d, J=12.1 Hz, 1H); 6.89 (d,J=6.3 Hz, 1H); 7.25 (t, J=7.8 Hz, 1H); 7.50 (m, 1H); 7.61 (m, 1H); 8.04(d, J=8.9 Hz, 1H)

C031—TFA Salt

LC-MS: t_(R) (min)=0.84; [M+H]: m/z 532; [M−H]⁻: m/z 530 (method A).

¹H NMR (400 MHz, DMSO-d₆+TFA): 0.83 (s, 9H); 1.11 (d broad, J=15.2 Hz, 1H); 1.22 (s, 3H); 1.83 (m, 1H); 2.00 to 2.36 (m, 4H); 3.82 (m, 1H); 4.20(dd, J=2.9 and 7.7 Hz, 1H); 4.36 (d, J=10.5 Hz, 1H); 5.00 (d, J=10.5 Hz,1H); 6.53 to 6.74 (m, 2H); 6.94 (dd, J=5.6 and 8.8 Hz, 1H); 7.21 (t,J=8.0 Hz, 1H); 7.41 (t, J=8.0 Hz, 1H); 7.71 (t, J=8.0 Hz, 1H).

C03401—TFA Salt(2′S,3′R,4′R,5′R)-6-Chloro-4′-(3-chloro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.58 (d, J=8.07 Hz, 1H), 7.30-7.10 (m, 4H),7.02 (d, J=7.67 Hz, 1H), 6.77 (d, J=1.54 Hz, 1H), 5.40-5.20 (m, 1H),4.44 (d, J=7.09 Hz, 1H), 4.10 (d, J=11.25 Hz, 1H), 3.95-3.80 (m, 1H),2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.85 (m, 2H), 1.80-1.70 (m,1H), 1.27 (s, 3H), 1.20-1.10 (m, 1H), 0.86 (s, 9H); ¹³C NMR (75 MHz,MeOH-d₄): 177.8, 167.3, 145.3, 137.1, 135.8, 134.4, 131.4, 130.4, 129.5,128.3, 126.3, 124.2, 124.1, 112.2, 67.3, 64.9, 64.2, 62.8, 57.2, 45.7,45.6, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₄³⁵Cl₂N₃O₃ [M+H]⁺: 530.1977. Found: 530.58.

LC-MS: t_(R) (min)=0.84; [M+H]⁺: m/z 530; [M−H]⁻: m/z 528 (method A).

¹H NMR (400 MHz, DMSO-d₆+TFA): 0.80 (s, 9H); 1.02 (d broad, J=15.5 Hz, 1H); 1.20 (s, 3H); 1.68 (m, 1H); 1.82 to 2.00 (m, 2H); 2.09 (m, 1H); 2.27(m, 1H); 3.73 (m, 1H); 4.07 (d, J=11.9 Hz, 1H); 4.32 (dd, J=2.2 and 8.4Hz, 1H); 5.29 (d, J=11.9 Hz, 1H); 6.74 (d, J=2.0 Hz, 1H); 6.98 (d, J=7.8Hz, 1H); 7.15 to 7.35 (m, 4H); 7.78 (t, J=8.3 Hz, 1H).

C03701—TFA Salt(2′S,3′R,4′R,5′R)-6-Chloro-4′-(3-chloro-phenyl)-2′-(2,2-dimethyl-propyl)-5-fluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 9.00-8.80 (m, 1H), 7.73 (d, J=8.42 Hz, 1H),7.40-7.20 (m, 3H), 7.15-7.05 (m, 1H), 6.89 (d, J=6.00 Hz, 1H), 5.32 (d,J=11.34 Hz, 1H), 4.52 (d, J=7.91 Hz, 1H), 4.20 (d, J=11.28 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.20-1.90 (m,2H), 1.90-1.80 (m, 1H), 1.31 (s, 3H), 1.30-1.15 (m, 1H), 0.91 (s, 9H);¹³C NMR (75 MHz, MeOH-d₄): 178.8, 168.2, 157.3 (d, J_(C-F)=255.8 Hz),142.1 (d, J_(C-F)=2.6 Hz), 137.1, 135.5, 132.8, 131.7, 130.7, 129.6,127.2 (d, J_(C-F)=7.2 Hz), 124.7 (d, J_(C-F)=19.3 Hz), 115.5 (d,J_(C-F)=24.9 Hz), 114.8, 68.6, 66.6, 65.3, 64.0, 58.2, 47.0, 46.8, 44.7,39.5, 32.2, 30.8, 28.8; ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺:548.1883. Found: 548.42.

LC-MS: t_(R) (min)=0.87; [M+H]⁺: m/z 548; [M−H]⁻: m/z 546 (method A).

¹H NMR (400 MHz, DMSO-d₆): 0.70 to 2.23 (m, 18H); 3.68 to 5.10 (m, 4H);6.78 to 8.03 (m, 6H).

C04801—TFA Salt(2′S,3′R,4′S,5′R)-6-Chloro-4′-(2,3-difluoro-phenyl)-2′-(2,2-dimethyl-propyl)-5-fluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (3-hydroxy-3-methyl-cyclobutyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 9.00-8.80 (m, 1H), 7.70 (d, J=8.35 Hz, 1H),7.50-7.35 (m, 1H), 7.30-7.10 (m, 2H), 6.88 (d, J=6.88 Hz, 1H), 5.30 (d,J=11.32 Hz, 1H), 4.66 (d, J=11.33 Hz, 1H), 4.56 (d, J=7.43 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m,2H), 1.80-1.70 (m, 1H), 1.30 (s, 3H), 1.16 (d, J=15.34 Hz, 1H), 0.90 (s,9H); ¹³C NMR (75 MHz, MeOH-d₄): 177.5, 166.9, 160-145 (m, 2×C_(sp2)-F),155.9 (d, J_(C-F)=243.4 Hz), 140.7 (d, J_(C-F)=2.69 Hz, 1H), 126.5-126.1(m), 125.6 (d, J_(C-F)=7.6 Hz), 125.0 (d, J_(C-F)=3.4 Hz), 123.6 (d,J_(C-F)=19.5 Hz), 122.0 (d, J_(C-F)=9.8 Hz), 119.1 (d, J_(C-F)=17.1 Hz),114.7 (d, J_(C-F)=25.0 Hz), 113.4, 67.3, 64.7, 64.3, 62.5, 48.2, 45.6,45.6, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₂³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.42.

LC-MS: t_(R) (min)=0.89; [M+H]⁺: m/z 550; [M−H]⁻: m/z 548 (method A)

¹H NMR (400 MHz, DMSO-d₆+TFA): 0.81 (s, 9H); 1.02 (d broad, J=15.5 Hz,1H); 1.20 (s, 3H); 1.62 (m, 1H); 1.87 to 1.98 (m, 2H); 2.09 (m, 1H);2.26 (m, 1H); 3.73 (m, 1H); 4.56 (m, 2H); 5.29 (d, J=12.4 Hz, 1H); 6.89(d, J=6.2 Hz, 1H); 7.20 to 7.49 (m, 3H); 8.06 (d, J=9.3 Hz, 1H).

MI-710201—TFA Salt(2′S,3′R,4′S,5′R)-6-Chloro-4′-(2,3-difluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.57 (d, J=8.0 Hz, 1H), 7.50-7.36 (m, 1H),7.27-7.07 (m, 3H), 6.79 (s, 1H), 5.11 (d, J=11.1 Hz, 1H), 4.55 (d,J=11.0 Hz, 1H), 4.39 (d, J=7.7 Hz, 1H), 3.71-3.52 (m, 1H), 3.52-3.37 (m,1H), 3.21 (dd, J=7.4, 14.5 Hz, 1H), 1.92 (d, J=9.6 Hz, 1H), 1.86-1.70(m, 2H), 1.58 (d, J=11.8 Hz, 1H), 1.43-1.18 (m, 4H), 1.12 (d, J=15.5 Hz,1H), 0.99 (d, J=13.0 Hz, 1H), 0.88 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58; HPLC (Condition I)t_(R)=52.15 min (Purity 98.8%).

LC-MS: t_(R) (min)=0.84; [M+H]⁺: m/z 546; [M−H]⁻: m/z 544 (method A).

¹H NMR (400 MHz; DMSO-d₆): mixture of isomers: 0.80 (s, 9H); 0.84 to1.30 (m, 5H); 1.41 to 1.87 (m, 5H); 3.43 to 3.54 (m, 2H); 4.03 (m broad,1H); 4.36 (d broad, J=10.3 Hz, 2H); 4.83 (m broad, 1H); 6.72 (d, J=2.0Hz, 1H); 7.10 (dd, J=2.0 and 8.3 Hz, 1H); 7.14 (m, 1H); 7.24 (m, 1H);7.40 (m, 1H); 7.58 (d, J=8.3 Hz, 1H); 7.97 (m broad, 1H); 10.56 (mbroad, 1H).

MI-710401—TFA Salt(2′S,3′R,4′S,5′R)-4′-(3-Chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-5,6-difluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.67 (t, J=8.6 Hz, 1H), 7.57 (t, J=6.8 Hz,1H), 7.38 (t, J=6.8 Hz, 1H), 7.15 (t, J=7.8 Hz, 1H), 6.71 (dd, J=6.6,10.1 Hz, 1H), 5.01 (d, J=10.8 Hz, 1H), 4.52 (d, J=10.8 Hz, 1H),4.40-4.21 (m, 1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.08-1.87 (m,2H), 1.87-1.68 (m, 2H), 1.68-1.53 (m, 1H), 1.45-1.18 (m, 3H), 1.17-0.97(m, 2H), 0.89 (s, 9H); ESI-MS calculated for C₂₉H₃₄ClF₃N₃O₃ (M+H)⁺requires 564.22. found 564.58; HPLC (Condition I) t_(R)=52.15 min(Purity 98.8%).

LC-MS: t_(R) (min)=0.84; [M+H]⁺: m/z 564; [M−H]⁻: m/z 562 (method A)

¹H NMR (400 MHz; DMSO-d₆+TFA): 0.81 (s, 9H); 0.81 to 1.98 (m, 10H); 3.35(m, 1H); 3.52 (m, 1H); 4.53 (d, J=12.2 Hz, 1H); 4.59 (dd, J=2.3 and 8.7Hz, 1H); 5.33 (d, J=12.2 Hz, 1H); 6.78 (dd, J=6.8 and 10.3 Hz, 1H); 7.21(t, J=9.0 Hz, 1H); 7.48 (m, 1H); 7.66 (m, 1H); 8.00 (m, 1H).

MI-710501—TFA Salt(2′S,3′R,4′S,5′R)-4′-(3-Chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-6-fluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.66-7.53 (m, 1H), 7.44-7.33 (m, 1H),7.22-7.09 (m, 1H), 6.93-6.79 (m, 1H), 6.59-6.51 (m, 1H), 5.40-5.31 (m,1H), 4.63-4.48 (m, 1H), 4.41-4.30 (m, 1H), 2.41-2.20 (m, 2H), 2.15-1.97(m, 2H), 1.95-1.85 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.47 (m, 3H),1.19-1.07 (m, 1H), 0.88 (s, 9H); ESI-MS calculated for C₂₉H₃₄ClF₃N₃O₃(M+H)⁺ requires 546.23. found 546.58; HPLC (Condition I) t_(R)=52.05 min(Purity 98.8%); HPLC (Condition II) t_(R)=19.26 min (Purity 100%).

LC-MS: t_(R) (min)=0.80; [M+H]: m/z 546; [M−H]⁻: m/z 544 (method A).

¹H NMR (400 MHz; DMSO-d₆+TFA): 0.81 (s, 9H); 0.81 to 1.99 (m, 10H); 3.35(m, 1H); 3.52 (m, 1H); 4.47 (d, J=12.0 Hz, 1H); 4.53 (d broad, J=8.3 Hz,1H); 5.37 (d, J=12.0 Hz, 1H); 6.58 (dd, J=2.6 and 9.1 Hz, 1H); 6.91 (m,1H); 7.21 (t, J=9.0 Hz, 1H); 7.45 (m, 1H); 7.62 to 7.72 (m, 2H).

MI-710601—TFA Salt(2′S,3′R,4′R,5′R)-6-Chloro-4′-(3-chloro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide, trifluoroacetate

LC-MS: t_(R) (min)=0.82; [M+H]⁺: m/z 544; [M−H]⁻: m/z 542 (method A).

¹H NMR (400 MHz; DMSO-d₆+TFA): 0.80 (s, 9H); 0.83 to 1.96 (m, 10H); 3.34(m, 1H); 3.50 (m, 1H); 4.02 (d, J=12.1 Hz, 1H); 4.48 (d broad, J=8.5 Hz,1H); 5.32 (d, J=12.1 Hz, 1H); 6.75 (dd, J=1.9 and 8.3 Hz, 1H); 6.98 (d,J=7.8 Hz, 1H); 7.18 to 7.32 (m, 4H); 7.74 (d, J=8.3 Hz, 1H).

MI-710801(2′S,3′R,4′R,5′R)-6-Chloro-4′-(3-chloro-phenyl)-2′-(2,2-dimethyl-propyl)-5-fluoro-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide

LC-MS: t_(R) (min)=0.85; [M+H]⁺: m/z 562; [M−H]⁻: m/z 560 (method A).

¹H NMR (400 MHz; DMSO-d₆): 0.78 (m, 1H); 0.81 (s, 9H); 1.08 to 1.33 (m,5 H); 1.70 to 1.90 (m, 4H); 3.25 (m, 1H); 3.35 to 3.58 (m, 3H); 3.92 (d,J=9.2 Hz, 1H); 4.43 (t, J=9.2 Hz, 1H); 4.49 (d, J=4.8 Hz, 1H); 6.79 (d,J=6.4 Hz, 1H); 6.92 (d, J=7.8 Hz, 1H); 7.09 to 7.21 (m, 3H); 7.75 (d,J=8.5 Hz, 1H); 7.83 (d, J=9.3 Hz, 1H); 10.38 (s broad, 1H).

MI-710901—TFA Salt(2′S,3′R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid cyclopropylamide, trifluoroacetate

¹H NMR (300 MHz, MeOH-d₄): 7.61 (d, J=8.1 Hz, 1H), 7.53 (t, J=6.7 Hz,1H), 7.40 (t, J=7.0 Hz, 1H), 7.21-7.08 (m, 2H), 6.79 (d, J=1.6 Hz, 1H),5.14 (d, J=11.3 Hz, 1H), 4.60 (d, J=11.3 Hz, 1H), 4.48 (d, J=7.0 Hz,1H), 2.78-2.58 (m, 1H), 1.86 (dd, J=8.4, 15.4 Hz, 1H), 1.13 (d, J=15.4Hz, 1H), 0.88 (s, 9H), 0.78-0.60 (m, 2H), 0.47-0.16 (m, 2H); ¹³C NMR (75MHz, MeOH-d₄): 177.9, 169.5, 157.8 (d, J_(C-F)=249.4 Hz), 145.2, 137.2,132.6, 128.7, 126.8 (d, J_(C-F)=1.6 Hz), 126.6 (d, J_(C-F)=4.9 Hz),124.2, 123.6, 122.5 (d, J_(C-F)=18.8 Hz), 121.8 (d, J_(C-F)=13.1 Hz),64.7, 64.4, 62.9, 43.5, 31.0, 29.6, 23.9, 6.7, 6.5; ESI-MS calculatedfor C₂₆H₂₉Cl₂FN₃O₂ (M+H)⁺ requires 504.16. found 504.58; HPLC (ConditionI) t_(R)=58.22 min (Purity 99.6%).

LC-MS: t_(R) (min)=0.98; [M+H]⁺: m/z 504; [M−H]⁻: m/z 502 (method A)

¹H NMR (400 MHz; DMSO-d₆+TFA): 0.15 (m, 1H); 0.35 (m, 1H); 0.54 to 0.70(m, 2H); 0.81 (s, 9H); 1.01 (d, J=15.2 Hz, 1H); 1.91 (dd, J=8.4 and 15.2Hz, 1H); 2.66 (m, 1H); 4.52 (m, 2H); 5.27 (d, J=12.0 Hz, 1H); 6.79 (d,J=2.0 Hz, 1H); 7.18 (dd, J=2.0 and 8.3 Hz, 1H); 7.23 (d, J=8.1 Hz, 1H);7.48 (m, 1H); 7.59 (m, 1H); 7.74 (d, J=8.3 Hz, 1H).

C08301—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 7.64-7.54 (m, 1H), 7.20-7.46 (m, 1H),7.46-7.38 (m, 1H), 7.28-7.14 (m, 2H), 5.26 (d, J=11.34 Hz, 1H), 4.65 (d,J=11.43 Hz, 1H), 4.55 (dd, J=8.28, 1.59 Hz, 1H), 3.95-3.80 (m, 1H),2.46-2.33 (m, 1H), 2.30-2.20 (m, 1H), 2.40-1.84 (m, 2H), 1.76-1.64 (m,1H), 1.29 (s, 3H), 1.17 (dd, J=15.40, 1.5 Hz, 1H), 0.88 (s, 9H); ¹³C NMR(75 MHz, MeOH-d₄): 177.4, 166.9, 157.8 (d, J_(C-F)=249.9 Hz), 144.3 (d,J_(C-F)=248.0 Hz), 132.8, 132.5 (d, J_(C-F)=12.3 Hz), 128.6, 126.7 (d,J_(C-F)=4.87 Hz), 126.3 (d, J_(C-F)=3.32 Hz), 125.6, 123.9 (d,J_(C-F)=14.3 Hz), 122.6 (d, J_(C-F)=18.9 Hz), 122.1, 121.3 (d,J_(C-F)=13.1 Hz), 67.2, 64.7, 64.5, 62.6, 48.9, 45.6, 45.5, 43.4, 38.3,31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₂₃₅Cl₂F2N₃O₃[M+H]⁺: 566.18.Found: 566.50.

C08601-TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.83 (d, J=6.98 Hz, 1H), 7.54-7.38 (m, 2H),7.30-7.14 (m, 3H), 5.26 (d, J=11.37 Hz, 1H), 4.64 (d, J=11.37 Hz, 1H),4.56 (dd, J=8.35, 1.27 Hz, 1H), 3.95-3.80 (m, 1H), 2.48-2.34 (m, 1H),2.32-2.20 (m, 1H), 2.08-1.86 (m, 2H), 1.80-1.64 (m, 1H), 1.30 (s, 3H),1.18 (d, J=14.37 Hz, 1H), 0.90 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄):177.4, 166.9, 133.9, 132.4, 130.1, 126.35 (dd, J_(C-F)=9.43, 2.72 Hz),125.5, 125.0 (d, J_(C-F)=3.72 Hz), 124.0 (d, J_(C-F)=14.4 Hz), 122.1 (d,J_(C-F)=3.13 Hz), 121.9 (d, J_(C-F)=9.77 Hz), 119.3 (d, J_(C-F)=17.1Hz), 119.9, 115.1, 67.3, 64.8, 64.7, 64.5, 62.6, 45.6, 45.5, 43.4, 38.4,31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₂ ³⁵ClF₃N₃O₃ [M+H]⁺:550.20. Found: 550.35.

C09101-TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.43 (d, J=7.73 Hz, 1H), 7.66-7.54 (m, 2H),7.39 (t, J=7.31 Hz, 1H), 7.24-7.10 (m, 2H), 6.82 (d, J=1.43 Hz, 1H),5.26 (d, J=12.22 Hz, 1H), 4.61 (d, J=11.38 Hz, 1H), 4.53 (d, J=7.73 Hz,1H), 3.57-3.54 (m, 1H), 1.93 (dd, J=15.41, 8.26 Hz, 1H), 1.80-1.10 (m,9H), 1.16 (s, 3H), 0.89 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 177.6,166.6, 157.7 (d, J_(C-F)=249.6 Hz), 144.9, 137.0, 132.4, 128.6, 126.5,126.4 (d, J_(C-F)=4.9 Hz), 124.0, 123.3, 122.3 (d, J_(C-F)=19.2 Hz),121.3 (d, J_(C-F)=13.0 Hz), 118.7, 114.9, 112.0, 68.7, 64.4, 64.0, 62.8,50.2, 48.9, 43.2, 37.9, 37.8, 30.9, 30.8, 29.4, 28.5, 28.3; ESI-MScalculated for C₃₀H₃₇ ³⁵Cl₂FN₃O₃ [M+H]⁺: 576.21. Found: 576.58.

C09601-TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.84 (d, J=6.87 Hz, 1H), 7.62 (d, J=8.10 Hz,1H), 7.19 (dd, J=8.10, 1.79 Hz, 1H), 7.15 (dt, J=8.42, 1.87 Hz, 1H),7.01 (s, 1H), 6.94 (d, J=9.62, 1.30 Hz, 1H), 6.83 (d, J=1.69 Hz, 1H),5.21 (d, J=11.25 Hz, 1H), 4.45 (dd, J=8.21, 1.56 Hz, 1H), 4.15 (d,J=11.25 Hz, 1H), 4.00-3.82 (m, 1H), 2.50-2.36 (m, 1H), 2.36-2.22 (m,1H), 2.10-1.98 (m, 1H) 1.91 (dd, J=15.44, 8.30 Hz, 1H), 1.84-1.72 (m,1H), 1.31 (s, 3H), 1.16 (dd, J=15.44, 1.47 Hz, 1H), 0.90 (s, 9H); ¹³CNMR (75 MHz, MeOH-d₄): 177.7, 167.0, 164.0 (d, J_(C-F)=249.6 Hz), 145.3,137.3, 136.6 (d, J_(C-F)=20.6H), 136.6, 126.4, 126.1 (d, J_(C-F)=2.91Hz), 124.3, 123.8, 117.9 (d, J_(C-F)=25.0 Hz), 115.4 (d, J=23.0 Hz),112.3, 67.3, 64.8, 64.3, 62.7, 56.7, 45.7, 45.6, 43.3, 38.4, 31.0, 29.6,27.6; ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺: 548.18. Found:548.67.

C09701—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.21 (d, J=6.81 Hz, 1H), 7.68-7.54 (m, 2H),7.39 (td, J=7.60, 1.36 Hz, 1H), 7.18 (t, J=8.09 Hz, 1H), 7.13 (dd,J=7.97, 1.92 Hz, 1H), 6.79 (d, J=1.66 Hz, 1H), 5.30 (d, J=11.51 Hz, 1H),4.56 (d, J=11.84 Hz, 1H), 4.52 (d, J=8.26 Hz, 1H), 3.86-3.70 (m, 1H),2.00-1.80 (m, 2H), 1.65-1.05 (m, 8H), 1.04 (s, 3H), 0.87 (s, 9H); ¹³CNMR (75 MHz, MeOH-d₄): 177.8, 167.2, 145.1, 137.2, 132.7, 128.8, 126.7(d, J_(C-F)=3.6 Hz), 126.6, 124.2, 123.4, 122.6 (d, J_(C-F)=18.7 Hz),121.7 (d, J_(C-F)=12.5 Hz), 112.1, 69.7, 64.6, 64.2, 62.9, 49.2, 43.4,36.8, 36.4, 31.0, 29.5, 28.3, 28.3; ESI-MS calculated for C₃₀H₃₇³⁵Cl₂FN₃O₃ [M+H]⁺: 576.21. Found: 576.67.

C11701—TFA Salt

¹H NMR (300 MHz, MeOH-d₄): 8.83 (d, J=6.61 Hz, 1H), 7.64 (dd, J=6.06,2.50 Hz, 1H), 7.59 (d, J=8.11 Hz, 1H), 7.36-7.22 (m, 1H), 7.11 (dd,J=8.09, 1.76 Hz, 1H), 7.00-6.82 (m, 1H), 1.72 (d, J=1.72 Hz, 1H), 5.13(d, J=11.21 Hz, 1H), 4.58 (d, J=11.21 Hz, 1H), 4.51 (dd, J=8.23, 1.66Hz, 1H), 3.98-3.80 (m, 1H), 2.44-2.32 (m, 1H), 2.32-2.20 (m, 1H),2.10-1.94 (m, 1H), 1.88 (dd, J=15.38, 8.24 Hz, 1H), 1.80-1.68 (m, 1H),1.29 (s, 3H), 1.14 (dd, J=15.38, 1.45 Hz, 1H), 0.89 (s, 9H); ¹³C NMR (75MHz, MeOH-d₄): 177.9, 167.1, 161.1 (d, J_(C-F)=243.6 Hz), 145.2, 137.3,136.9, 132.1 (d, J_(C-F)=9.0 Hz), 131.2 (d, J_(C-F)=3.60 Hz), 130.0 (d,J_(C-F)=2.22 Hz), 127.0, 123.8 (d, J_(C-F)=57.7 Hz), 121.6 (d,J_(C-F)=14.8 Hz), 118.5 (d, J_(C-F)=25.4 Hz), 112.1, 67.4, 64.5, 64.4,62.7, 48.4, 45.7, 45.5, 43.3, 38.4, 31.0, 29.6, 27.6; ESI-MS calculatedfor C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺: 548.19. Found: 548.67.

C29701—TFA Salt

¹H NMR (MeOH-d₄): 7.66 (d, J=8.47 Hz, 1H), 7.56 (t, J=6.87 Hz, 1H), 7.43(td, J=7.60, 1.47 Hz, 1H), 7.22 (d, J=7.79 Hz, 1H), 7.16 (dd, J=8.17,1.89 Hz, 1H), 6.81 (d, J=1.80 Hz, 1H), 5.17 (d, J=11.92 Hz, 1H),4.68-4.58 (m, 2H), 3.91-3.78 (m, 1H), 3.78-3.66 (m, 2H), 2.44-2.32 (m,1H), 2.26-2.10 (m, 2H), 1.98-1.88 (m, 1H), 1.64-1.52 (m, 1H), 1.43 (t,J=7.15 Hz, 1H), 1.33-1.25 (m, 1H), 1.29 (s, 3H), 0.81 (s, 3H); ESI-MS:Calculated for C₃₀H₃₇Cl₂FN₃O₃ [M+H]⁺=576.22. Found: 576.92.

C30201—TFA Salt

¹H NMR (MeOH-d₄): 7.68 (dd, J=8.16, 1.47 Hz, 1H), 7.48 (t, J=7.09 Hz,1H), 7.39 (td, J=7.30, 1.29 Hz, 1H), 7.20-7.08 (m, 2H), 6.79 (d, J=1.79Hz, 1H), 5.19 (d, J=11.56 Hz, 1H), 4.83 (d, J=11.56 Hz, 1H), 4.51 (t,J=3.96 Hz, 1H), 3.94-3.78 (m, 1H), 3.12 (s, 3H), 2.50-2.36 (m, 1H),2.32-2.20 (m, 1H), 2.10-1.92 (m, 2H), 1.73 (dd, J=10.67, 9.23 Hz, 1H),1.40-1.25 (m, 1H), 1.29 (s, 3H), 0.75 (s, 9H); ESI-MS: Calculated forC₂₉H₃₅Cl₂FN₃O₃ [M+H]⁺=562.20. Found: 562.58.

Example 2 Fluorescence-Polarization MDM2 Binding Assay

The binding affinity of the MDM2 inhibitors was determined using anoptimized, sensitive and quantitative fluorescence polarization-based(FP-based) binding assay using a recombinant human His-tagged MDM2protein (residues 1-118) and a fluorescently tagged p53-based peptide.

The design of the fluorescence probe was based upon a previouslyreported high-affinity p53-based peptidomimetic compound(5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH₂ (SEQID NO: 1)) (García-Echeverría et al., J. Med. Chem. 43: 3205-3208(2000)). This tagged peptide is called PMDM6-F. The K_(d) value ofPMDM6-F with the recombinant MDM2 protein was determined from thesaturation curve. MDM2 protein was serially double diluted in a Dynex96-well, black, round-bottom plate, and the PMDM6-F peptide was added at1 nM concentration. The assay was performed in the buffer: 100 mMpotassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02%sodium azide, 0.01% Triton X-100) and the polarization values weremeasured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc.,Research Triangle Park, N.C.). The IC₅₀ value was obtained by fittingthe mP values in a sigmoidal dose-response curve (variable slope) with anon-linear regression, and was determined to be 1.40 nM±0.25. The K_(d)value was calculated using the equation: K_(d) value=IC₅₀−L0/2. L0 isthe total concentration of the fluorescent ligand; L0/2 is the totalconcentration of the fluorescent ligand divided by 2. Since PMDM6-F wasused at a final concentration of 1 nM, L0/2 was 0.5 nM.

Dose-dependent, competitive binding experiments were performed withserial dilutions of a tested compound in DMSO. A 5 μL sample of thetested compound and pre-incubated MDM2 protein (10 nM) and PMDM6-Fpeptide (1 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5;100 μg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% TritonX-100), were added in a Dynex 96-well, black, round-bottom plate toproduce a final volume of 125 μL. For each assay, the controls includedthe MDM2 protein and PMDM6-F (equivalent to 0% inhibition), PMDM6-Fpeptide alone (equivalent to 100% inhibition). The polarization valueswere measured after 3 h of incubation. The IC₅₀ values, i.e. theinhibitor concentration at which 50% of bound peptide is displaced, weredetermined from a plot using nonlinear least-squares analysis. Curvefitting was performed using GRAPHPAD PRISM software (GraphPad Software,Inc., San Diego, Calif.).

In the alternative, fluorescence polarization values were measured usingthe Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park,N.C.) in Microfluor 2 96-well, black, round-bottom plates (ThermoScientific). In the saturation experiments, 1 nM of PMDM6-F andincreasing concentrations of proteins were added to each well to a finalvolume of 125 μl in the assay buffer (100 mM potassium phosphate, pH7.5, 100 μg/ml bovine γ-globulin, 0.02% sodium azide (Invitrogen), with0.01% Triton X-100 and 4% DMSO). Plates were mixed and incubated at roomtemperature for 30 minutes with gentle shaking to assure equilibrium.The polarization values in millipolarization units (mP) were measured atan excitation wavelength of 485 nm and an emission wavelength of 530 nm.Equilibrium dissociation constants (K_(d)) were then calculated byfitting the sigmoidal dose-dependent FP increases as a function ofprotein concentrations using Graphpad Prism 5.0 software (GraphpadSoftware, San Diego, Calif.).

K_(i) values of tested compounds were determined in a dose-dependentcompetitive binding experiment. Mixtures of 5 μl of the tested compoundin different concentrations in DMSO and 120 μl of preincubatedprotein/fluorescent probe complex with fixed concentrations in the assaybuffer (100 mM potassium phosphate, pH 7.5, 100 μg/ml bovine γ-globulin,0.02% sodium azide, with 0.01% Triton X-100) were added into assayplates and incubated at room temperature for 30 minutes with gentleshaking. Final concentrations of the protein and fluorescent probe inthe competitive assays were 10 nM and 1 nM, respectively, and final DMSOconcentration is 4%. Negative controls containing protein/fluorescentprobe complex only (equivalent to 0% inhibition), and positive controlscontaining free fluorescent probe only (equivalent to 100% inhibition),were included in each assay plate. FP values were measured as describedabove. IC₅₀ values were determined by nonlinear regression fitting ofthe sigmoidal dose-dependent FP decreases as a function of totalcompound concentrations using Graphpad Prism 5.0 software (GraphpadSoftware, San Diego, Calif.). K_(i) values of tested compounds to theMDM2 protein were calculated using the measured IC₅₀ values, the K_(d)value of the fluorescent probe to the protein, and the concentrations ofthe protein and fluorescent probe in the competitive assays(Nikolovska-Coleska et al., Anal. Biochem. 332:261-73 (2004)).

Compounds shown in Table 2A as the free base were tested either as thefree base or as the CF₃CO₂H (TFA) or HCl salt. In general, comparableassay responses are expected between the free base and salt form of acompound (See, e.g., MI-77301 (free base) and MI-77301 (TFA salt)).

Binding Kinetics of Different Isomers in Binding Media

Aliquots of freshly prepared DMSO stock solutions of compounds werediluted in FP binding assay buffer to prepare the aqueous compoundincubation solutions in which compound isomerization was taking place.Final compound concentration in the incubation solution was 25 μM, and5% of DMSO was present to enhance the solubility. These solutions werestored at room temperature for the whole time range of the experiment.80 μL of aliquots of compound solutions were mixed with 20 μL of freshlyprepared MDM2/PMDM6-F mixture in the assay plates at different timepoints. Final concentrations of the protein, fluorescent probe, and DMSOare same as those in the competitive assays described above. Negativeand positive controls were included in each assay plate as well.Following 15 minutes of incubation at room temperature with gentleshaking, mP values were measured and IC₅₀ values were determined asdescribed above (Table 2B). Due to the plate preparation and incubationtime required before measurement, it should be noticed that all IC₅₀values presented below are values actually obtained 20 minutes after thelabeled incubation time.

Example 3 Fluorescence-Polarization MDM2 Binding Assay

The binding affinity of the MDM2 inhibitors was optionally determinedusing a fluorescence polarization-based (FP-based) binding assay using arecombinant human MDM2 protein (residues 5-109) and PMDM6-F as follows:

MDM2 protein was serially diluted with a step of 1.8 in a Costar96-well, black, non binding surface reference 3686 plate, and thePMDM6-F peptide was added at 5 nM concentration. The assay was performedin the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovinegamma globulin, 0.01% Triton X-100) and the anisotropy values weremeasured at equilibrium using a Fusion reader (Packard). The fraction ofligand bound, F_(SB), was calculated using the following equationF_(SB)=(Aobs−AF)/[(Ab−Aobs)Q+Aobs−AF](ref) where Aobs=anisotropyobserved, Ab=anisotropy when all p53 is bound, AF=anisotropy when p53 isfree, Q=ratio Fluorescence intensity Bound/fluorescence intensity Free(Biochemistry 43:16056-16066 (2004)). KD was determined, using theLangmuir equation applied to fluorescence polarization, to be 1.8 nM.

Dose-dependent, competitive binding experiments were performed withserial dilutions of a tested compound in DMSO. A 5 μL sample of thetested compound and PMDM6-F peptide (5 nM) and MDM2 protein (6 or 8 nM)in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 μg/mLbovine gamma globulin, 0.01% Triton X-100), were added in Costar96-well, black, non binding surface reference 3686 to produce a finalvolume of 125 μL. For each assay, the controls included the MDM2 proteinand PMDM6-F (equivalent to 0% inhibition), PMDM6-F peptide alone(equivalent to 100% inhibition). The polarization values were measuredat equilibrium. The IC₅₀ values, i.e. the inhibitor concentration atwhich 50% of bound peptide is displaced, were determined from a plotusing the 4-parameter logistic model (Ratkowsky and Reedy, Biometrics42(3):575-82 (1986). The adjustment was obtained by non-linearregression using the Marquardt algorithm in Xlfit software (Table 3).

TABLE 3 Compound IC₅₀ (biot MDM2) IC₅₀ (without Tag) MI-710201 308.32 nM272.83 nM MI-710501 309.08 nM 260.40 nM MI-710601 157.86 nM 137.94 nMMI-710801 289.62 nM 256.02 nM MI-710901 224.96 nM 184.74 nM C02701149.49 nM 111.52 nM C02901 159.97 nM 121.90 nM C03001 120.23 nM  89.59nM C031 3339.44 nM  2742.86 nM  C03401 107.66 nM  85.61 nM C03701 270.09nM 220.41 nM C04801 473.01 nM 388.72 nM

Example 4 Cell Growth Assay

Isogenic HCT-116 colon cancer cell lines were a kind gift from Prof.Bert Vogelstein (Johns Hopkins, Baltimore, Md.) and were maintained inMcCoy's 5A medium containing 10% FBS. All other cell lines were obtainedfrom ATCC (Manassas, Va.) and were maintained in RPMI-1640 mediumcontaining 10% FBS.

Cells were seeded in 96-well flat bottom cell culture plates at adensity of 2-3×10³ cells/well with compounds and incubated for 4 days.The rate of cell growth inhibition after treatment with increasingconcentrations of the tested compounds was determined by WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt (Dojindo Molecular Technologies Inc., Gaithersburg,Md.). WST-8 was added at a final concentration of 10% to each well, andthen the plates were incubated at 37° C. for 2-3 hrs. The absorbance ofthe samples was measured at 450 nm using a TECAN ULTRA Reader. Theconcentration of the compounds that inhibited cell growth by 50% (IC₅₀)was calculated by comparing absorbance in the untreated cells and thecells treated with the compounds using the GraphPad Prism software(GraphPad Software, La Jolla, Calif. 92037, USA). The results of thisassay are presented in Tables 2A and 2C. Under conditions used in thisassay, it is possible that a compound having Formula II isomerizes to acompound having Formula XII and other isomers (e.g., MI-773 isomerizesto MI-77301; MI-519-64 isomerizes to MI-519-6401). The results of thisassay for MI-77301 in a variety of melanoma cell lines (Fernandez, Y.,et al., Cancer Res. 65:6294-6304 and references cited therein) ispresented in FIG. 24.

Example 5 Cell Death Assay

Cell death assays were performed using trypan blue staining. Cells weretreated in the presence and absence of indicated compounds. Both thefloating and adherent cells were stained with trypan blue. Cells thatstained blue or the morphologically unhealthy cells were scored as deadcells. At least 100 cells were counted in each of three separate areasunder microscope. As shown in FIGS. 11 and 12, MDM2 inhibitors providedherein induce cell death in SJSA-1 and RS4;11 cancer cells withwild-type p53.

TABLE 2A IC₅₀ (μM, unless otherwise indicated) SJSA-1 HCT-116 LNCAP PC-3Exam- (p53 wild- (p53 wild- (p53 wild- HCT-116 (deleted ple ChemicalStructure MDM2 type) type) type) (p53—/—) p53) MI- 219

180 nM MI- 21901

104 nM MI- 519-64

41.4 ± 2.6  nM 0.27¹ 0.18¹ 0.50¹ 13.69¹ 6.1¹ MI- 519-65

<1 <3 <3 <3 >10 NT MI- 773

62.1 ± 17.8 nM 0.061 0.058 0.085 13.3 14.7 MI- 786

<1 0.8 0.5 1.2 NT 14.4 C027

0.167 <3 <3 <3 >10 >10 C029

0.176 <3 <3 <3 >5 >5 C031

<5 <3 <3 <3 >10 >10 C034

0.048 <3 <3 <3 >10 >10 C035

<3 <5 <5 <5 >10 >10 C086

0.402 MI- 7102

175 ± 19  nM 0.42 0.20 0.25 26.8 19.1 MI- 7104

169 ± 30  nM 0.9 0.37 0.49 16.6 16.5 MI- 7105

520 ± 190 nM 0.26 0.28 0.18 18.7 21.3 MI- 791

<3 <3 <3 <3 >10 >10 MI- 519- 6401

5.6 ± 1.7 nM 0.40 ± 0.24¹  0.102 ± 0.052¹  0.33 ± 0.012¹ 12.2 ± 0.8¹10.51 ± 3.32¹ MI- 77301 (free amine)

8.2 ± 3.0 nM 0.058 ± 0.023¹ 0.033 ± 0.01¹ 0.054 ± 0.008¹  9.1 ± 1.7¹ 8.45 ± 0.35¹ MI- 77301 (TFA salt)

13.3 ± 2.6  nM 0.083; 0.075¹  0.048 ± 0.014¹ 0.086 ± 0.016¹ 12.9 ± 2.8¹13.0 ± 3.0¹ MI- 77302 (TFA salt)

5.9 C02701

0.027 0.2 0.2 0.3 13.2 12.9 C02901

0.035 0.5 0.6 0.2 16.4 15.6 C03001

<0.1 0.2 0.2 0.1 14.2 29.8 C03401

0.018 0.3 0.6 0.3 17.3 17.4 C03701

0.061 0.7 0.2 0.4 21.0 26.7 C08301

<0.1 0.2 0.3 10.1 C08601

0.162 C09101

0.008 C09601

0.012 MI- 710201

61.7 ± 5.1  nM 0.57 0.25 0.3 27.6 19.3 MI- 710301

35.4 0.13 0.92 0.10 11.9 12.50 MI- 710401

70.2 ± 14.1 nM 0.90 0.37 0.49 16.6 16.5 MI- 710601

20.1 ± 5.0  nM 0.38 0.26 0.21 11.5 18.4 MI- 710801

32.0 ± 5.9  nM C09701

0.011 0.1 0.1 0.2 10.6 11.1 C11701

0.159 1.4 1.5 21.8 19.6 CB061- Isomer B

<0.1 CB087- Isomer B

<0.5 CB083- Isomer B

>5 CB084- Isomer B

>5 C29701

<0.1 C30201

<0.1 ¹Four day treatment.

TABLE 2B IC₅₀ values (nM) to human Time MDM2 protein in FP assay (hours)MI-773 MI-77301 MI-519-64 MI-519-6401 0.1 79.9 11.3 77.2 6.5 3.0 74.311.2 54.0 7.0 7.0 65.2 12.3 46.2 8.8 24.0 57.7 15.1 43.4 8.9 32.0 49.210.6 42.7 8.3 48.0 46.6 14.5 34.8 8.8 72.0 33.3 12.8 27.9 9.6

TABLE 2C RS4; 11 (p53 wt) Example IC₅₀ (μM) MI-519-6401 0.13 ± 0.02¹MI-773  0.13¹ (TFA salt) MI-77301 0.102; 0.081¹ (free amine) MI-773010.059 ± 0.025¹ (TFA salt) MI-7102 0.46 MI-710201 0.54 MI-710301 0.16MI-7104 0.52 MI-710401 0.66 MI-7105 0.37 MI-710601 0.13 ¹Four daytreatment.

Example 6 Western Blotting

For Western blot analysis, cells were lyzed in ice-cold RIPA buffer: 20mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% sodiumdeoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mMsodium orthovanadate and 1 μg/ml leupeptin. The proteins in the wholecell lysates were detected by Western blot analysis using the followingantibodies: anti-p53 (clone DO-1), anti-MDM2 (clone SMP-14), anti-p21(clone SX118), anti-β-actin (clone AC-40) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH; HRP conjugated). As shown in FIGS. 8, 9, and13-16, MDM2 inhibitors provided herein are active in this assay.

PARP cleavage was used as a biochemical marker of apoptosis. Duringapoptosis, caspases cleave Poly(ADP-ribose)polymerase (PARP). Rabbitanti-PARP (Cell Signaling Cat #9542), used in the experiment, detectscleavage of full length (116 kD) Poly(ADP-ribose)polymerase (PARP) andlarger (89 kD) cleaved fragment of PARP. A total of 2×10⁶ adherent cellswere treated in the presence or absence of MI-77301 and incubated at 37°C. for 19 hr. Cells were harvested using 0.05% trypsin-EDTA(Invitrogen), washed in PBS and lysed on ice for 15 min using RIPAbuffer (Sigma), supplemented with protease inhibitor cocktail (Roche).Clarified cell lysate was obtained by centrifuging the lysed cells at13000×g at 4° C. for 15 min. Protein in the cell lysate was estimatedusing commercially available Bio-Rad protein assay dye. A total of 25 μgprotein was loaded on a 4-20% SDS-PAGE gel, electrophoresed, andtransferred to a PVDF membrane for 3 hours at 40 V. Membrane was blockedin TBST (20 mM Tris, 0.5 M NaCl, 0.1% Tween-20, pH 7.5) containing 5%dry milk (Bio-Rad) for 1 hr at room temperature. Primary antibodydiluted in TBST, containing 5% dry milk, was applied to the membraneovernight in cold room at 4° C. on an orbital shaker. Membrane waswashed in TBST, incubated for 1 hr at room temperature with either ananti-rabbit secondary antibody (Immunopure goat anti-rabbit antibody,Thermo Scientific) or an anti-mouse antibody (Pierce goat anti-mouseantibody, Thermo Scientific), diluted 1:2000 in TBST. Membrane waswashed in TBST and developed using SuperSignal West Pico reagent (ThermoScientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodyconjugated to HRP (Santa Cruz) was used as a loading control forproteins. Western blot analyses for MI-77301 are provided in FIGS. 25,26, and 28.

Example 7 In Vivo Efficacy Studies Using SJSA-1 and 22Rv1 XenograftModels

SJSA-1 (osteosarcoma) tumor cells were harvested with Trypsin(0.05%)-EDTA (0.53 mM) (GIBCO™, Invitrogen Corp.), growth medium addedand cells placed on ice. A cell sample was mixed 1:1 with Trypan Blue(GIBCO™, Invitrogen Corp.) and counted on a hemocytometer to determinethe number of live/dead cells. Cells were washed once with 1×PBS(GIBCO™, Invitrogen Corp.) and resuspended in PBS. For Matrigelinjections, after washing in PBS, cells are resuspended in an ice coldmixture of 1:1 PBS and Matrigel (BD Biosciences, Invitrogen Corp.) for afinal Matrigel protein concentration of 5 mg/ml. SJSA-1 tumors wereinoculated into C.B-17 SCID mice at 5×10⁶ cells in 0.1 ml with Matrigel.Cells were injected s.c. into the flank region of each mouse using a 27gauge needle.

The size of tumors growing in the mice was measured in two dimensionsusing calipers. Tumor volume (mm³)=(A×B²)/2 where A and B are the tumorlength and width (in mm), respectively. During treatment, tumor volumeand body weight was measured three times a week. After the treatment wasstopped, tumor volume (FIGS. 17, 20, 22, and 23) and body weight (FIGS.18 and 21) was measured at least once a week. Mice were kept for anadditional 60 days for further observation of tumor growth and toxicity.As shown in FIG. 22, a single 200 mg/kg dose of MI-77301 (QD1 treatment)shows comparable efficacy to a continuous dosing regimen (QD7treatment).

Before treatment began, tumors were allowed to grow to 60-140 mm³ involume, at which point the blood vessel supplies to the tumor shouldhave been established. Mice with tumors within acceptable size rangewere randomized into treatment groups of 8 mice for experimentalcompounds and 10 mice for the Control group. Experimental compounds weregiven orally, once per day for 2-3 weeks. The Control group receivedvehicle alone (10% PEG 400:3% Cremophor:87% PBS). Other suitablevehicles for in vivo administration of the compounds provided hereininclude, without limitation, 98% PEG 200:2% polysorbate 80; 98% PEG200:2% TPGS; and 0.5% polysorbate 80:0.6% methyl cellulose:98.9% water.

Using similar protocols, the antitumor activity of MI-519-6401 andMI-77301 was evaluated in the 22Rv1 prostate cancer model in mice (FIG.19), and the antitumor activity of MI-77301 was evaluated in the HCT-116human colorectal tumor model (FIG. 31), the LNCAP human prostate tumormodel (FIG. 32), and the RS4;11 human ALL model (FIG. 33).

Example 8 Synthesis of MI-519-64 and MI-519-65 Step 1: benzyl3-oxocyclobutanecarboxylate (2)

Referring to Scheme 6A, BnBr was added to the mixture of compound 1 andK₂CO₃ in acetonitrile 150 mL. The mixture was stirred at roomtemperature over 24 h and the solid was filtered. The solvent wasremoved and the residue was purified by column chromatography to givecompound 2.

Step 2: benzyl 3-hydroxy-3-methylcyclobutanecarboxylates (3 and 4)

MeMgCl in THF was added dropwise to the solution of compound 2 indiethyl ether at −78° C. and the mixture was stirred at the sametemperature for half an hour. After TLC monitoring showed thedisappearance of the starting material, the reaction was quenched byadding aqueous NH₄Cl solution. The aqueous phase was extracted withethyl acetate three times and the combined organic phase was washed withbrine and dried (Na₂SO₄). The solid was filtered and the solvent wasremoved. The residue was purified by column chromatography to givecompounds 3 and 4 (5:1 based on TLC analysis).

Step 3: benzyl 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanecarboxylates (5 and 6)

To the mixture of compounds 3 and 4 in DMF (10 mL) was added imidazoleand TBSCl, and the resulting mixture was stirred at 80° C. for 30 h.After cooling to room temperature, water was added and the aqueous phasewas extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried (Na₂SO₄). The solid was filtered and thesolvent was removed. The residue was purified by column chromatographyto get compounds 5 and 6.

Step 4: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanecarboxylicacids (7 and 8)

To the mixture of compounds 5 and 6 in isopropanol was added Pd/C. Theresulting mixture was stirred under 1 atm hydrogen for 1 h. TLC showedthe disappearance of the starting material and the solid was filtered.The solvent was removed to give compounds 7 and 8.

Step 5:benzyl-3-(tert-butyldimethylsilyloxy)-3-methylcyclobutylcarbamates 9 and10

To a 0° C. stirring solution of compounds 7 and 8 and Et₃N in acetonewas added ClCOOEt dropwise. The resulting mixture was stirred at 0° C.for 30 min. A solution NaN₃ in water was added, and the resultingmixture was stirred at 0° C. for an additional 20 min. Water was added,and the aqueous phase was extracted with ethyl acetate three times. Thecombined organic phase was washed with brine and dried (Na₂SO₄). Thesolvent was removed and the residue was dissolved in toluene. Benzylalcohol and NaHCO₃ were added. The resulting mixture was stirred at 80°C. for 2 h. All the solvent was removed and the residue was purified bycolumn chromatography to obtain two isomers 9 and 10 in a 5:1 ratio.

Step 6: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (11)

To a mixture of the major isomer 9 and NaHCO₃ in isopropanol was addedPd/C and the resulting mixture was stirred under 1 atm hydrogen for 1 h.The solid was filtered and the solvent was removed to give compound 11.

Step 7: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (12)

To a mixture of the minor isomer 10 and NaHCO₃ in isopropanol was addedPd/C and the resulting mixture was stirred under 1 atm hydrogen for 1 h.The solid was filtered and the solvent was removed to give compound 12.

Step 8: MI-519-64

Referring to Scheme 6B, to a solution of compound 11 in THF was addedcompound 13 and the resulting solution was stirred overnight. Thesolvent was removed and the residue thus obtained was dissolved inCH₃CN/H₂O (1:1). CAN was added and the reaction mixture was stirred for30 min. Water was added and the aqueous phase was extracted with ethylacetate three times. The combined organic layers were dried (Na₂SO₄),filtered, and concentrated. The residue was purified by columnchromatography on silica gel to give compound 14. Compound 14 wasdissolved in methanol, 12M HCl in water was added, and the reactionmixture was stirred for 1 h at room temperature. The solvent was removedand the residue was purified by HPLC to give MI-519-64 as the TFA salt.¹H NMR (300 MHz, CD₃OD) δ 7.54-7.52 (m, 1H), 7.42-7.38 (m, 1H),7.23-7.18 (m, 1H), 6.88-6.75 (m, 3H), 5.04 (d, J=9.9 Hz, 1H), 4.45 (d,J=9.9 Hz, 1H), 4.19-4.16 (m, 1H), 3.92-3.89 (m, 1H), 2.42-2.11 (m, 2H),2.10-1.87 (m, 3H), 1.32-1.24 (m, 4H), 0.82 (s, 9H); MS (ESI) m/z 548[M+H]⁺.

Step 9: MI-519-65

To a solution of compound 12 in THF was added compound 13 and theresulting solution was stirred overnight. The solvent was removed andthe residue was dissolved in CH₃CN/H₂O (1:1). CAN was added and thereaction mixture was stirred for 30 min. Water was added and the aqueousphase was extracted with ethyl acetate three times. The combined organiclayers were dried (Na₂SO₄), filtered, and concentrated. The residue waspurified by column chromatography on silica gel to give compound 15.Compound 15 was dissolved in methanol, 12M HCl in water was added, andthe reaction mixture was stirred for 1 h at room temperature. Thesolvent was removed and the residue was purified by HPLC to giveMI-519-65 as the TFA salt. ¹H NMR (300 MHz, CD₃₀D) δ 7.50 (m, 1H),7.44-7.38 (m, 1H), 7.24-7.20 (m, 1H), 6.89-6.88 (m, 1H), 6.80 (m, 1H),6.71 (m, 1H), 4.91-4.88 (m, 1H), 4.40-4.36 (m, 2H), 4.10-4.06 (m, 1H),2.41-2.33 (m, 2H), 2.07-1.87 (m, 3H), 1.25-1.21 (m, 4H), 0.82 (s, 9H);MS (ESI) m/z 548 [M+H]⁺.

Example 9 Synthesis of MI-519-6401

MI-519-64 (100 mg) purified by flash chromatography on silica gel wasplaced in a 50 mL round-bottom-flask equipped with magnetic stirringbar. Acetonitrile (20 mL) was added to fully dissolve the compound anddeionized water (7 to 10 mL) was added. NaHCO₃ saturated aqueoussolution (ca. 0.5 mL) was then added to adjust the pH value between 7and 8. This solution was allowed to stir at room temperature for atleast 12 h. TFA (0.1 mL) and another mL of deionized water were added tothe solution and the solution was purified by semi-preparative RP-HPLCimmediately using acetonitrile and water as the eluents to giveMI-519-6401 as the TFA salt. ¹H NMR (300 MHz, MeOH-d₄): 7.62-7.53 (m,2H), 7.45-7.35 (m, 1H), 7.20-7.10 (m, 2H), 6.80-6.85 (m, 1H), 5.11 (d,J=11.07 Hz, 1H), 4.57 (d, J=11.11 Hz, 1H), 4.40 (d, J=7.39 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m,1H), 1.90-1.60 (m, 2H), 1.30 (s, 3H), 1.20-1.05 (m, 1H), 0.88 (s, 9H);¹³C NMR (75 MHz, MeOH-d₄): 177.8, 168.0, 157.6 (d, J_(C-F)=249 Hz),144.9, 136.8, 132.2, 128.5, 126.5, 126.3 (d, J_(C-F)=4.76), 123.9,123.7, 122.3 (d, J_(C-F)=18.97 Hz), 122.0 (d, J_(C-F)=13.1 Hz), 111.8,67.1, 64.6, 64.5, 62.9, 49.0, 45.5, 45.4, 43.3, 38.0, 30.8, 29.5, 27.3;ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺: 548.1883. Found: 548.25.

Analytical RP-HPLC spectra are presented in FIGS. 1-3. Referring to FIG.3, MI-519-6401 corresponds to the RP-HPLC peak at 31.787 minutes.

In an alternate procedure, MI-519-64 (100 mg) purified by flashchromatography on silica gel was placed in a 50 mL round-bottom-flaskequipped with magnetic stirring bar. Methanol (20 mL) was added to fullydissolve the compound and deionized water (10 to 20 mL) was added.NaHCO₃ saturated aqueous solution (ca. 0.5 mL) was then added to adjustthe pH value between 7 and 8. This solution was allowed to stir at roomtemperature for at least 12 h. TFA (0.1 mL) and another 10 mL ofdeionized water were added to the solution and the solution was purifiedby semi-preparative RP-HPLC immediately using acetonitrile and water asthe eluents to give MI-519-6401 as the TFA salt.

C02701, C02901, C03001, C03401, C03701, C03801, C04801, C08301, C08601,and C11701 of EXAMPLE 1 were prepared using procedures similar to thatused to prepare MI-519-6401.

Example 10 Synthesis of MI-773

Step 1

To a stirred solution of oxindole 1 (4.19 g, 25 mmol) in methanol (50mL) was added aldehyde 2 (3.96 g, 25 mmol) and piperidine (2.45 mL, 25mmol). The reaction mixture was stirred at room temperature for 3 h andthe yellow precipitate was collected, washed successively with methanol,hexanes, and ethyl ether and dried to give compound 3 (6.25 g, 81%yield).

Step 2

To a solution of compound 3 (6.25 g, 21 mmol) in toluene (75 ml) wasadded compound 4 (5.43 g, 21 mmol), compound 5 (2.15 g, 21 mmol) and 4 Åmolecular sieves (4 g). The reaction mixture was heated at refluxovernight and filtrated. The filtrate was evaporated and the residue waspurified by silica gel flash column chromatography (n-hexane/ethylacetate=9:1 to 5:1) to give compound 6 (8.78 g, 65% yield).

Step 3

The solution of compound 6 (965 mg, 1.5 mmol) and amine 7 (346 mg, 3mmol) in 5 mL of THF was stirred at room temperature for 2 days and thesolvent was removed under reduced pressure. The residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=1:1 to1:4) to give compound 8 (819 mg, 72% yield).

Step 4

To an ice-bath cooled solution of compound 8 (800 mg, 1.05 mmol) inCH₃CN (8 ml), H₂O (4 ml) and acetone (4 ml) was added CAN (ammoniumcerium) (1.15 g, 2.1 mmol). Progress of the reaction was monitored byTLC. When all the starting material disappeared (around 5 min), 100 mgof NaHCO₃ powder was added and the reaction mixture was diluted with 50mL of ethyl acetate. The organic phase was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue was purified by silica gel flashcolumn chromatography (methylene chloride/methanol/triethylamine=200:1:1to 200:10:1) to give(2′R,3S,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide(MI-773) (402 mg, 68% yield, Purity (HPLC): >95% (FIG. 35)). Theabsolute stereochemical configuration of MI-773 was determined by x-rayanalysis.

MI-773 was dissolved in DCM, TFA was added, and the solvent was removedby evaporation. The residue was further purified by chromatography on aC18 reverse phase semi-preparative HPLC column with solvent A (0.1% ofTFA in water) and solvent B (0.1% of TFA in methanol) as eluents(gradient: 45% of solvent A and 55% of solvent B to 30% of solvent A and70% of solvent B in 30 min) to give MI-773 as the TFA salt. NMR forMI-773 (TFA salt): ¹H NMR (300 MHz, CD₃OD) δ 7.47 (t, J=7.0 Hz, 1H),7.34 (t, J=7.4 Hz, 1H), 7.14 (t, J=7.9 Hz, 1H), 6.83 (s, 1H), 6.80 (s,2H), 4.39 (d, J=10.0 Hz, 1H), 4.15-4.05 (m, 1H), 3.72-3.53 (m, 1H),3.53-3.85 (m, 2H), 2.10-1.75 (m, 4H), 1.62 (d, J=12.2 Hz, 1H), 1.45-1.05(m, 5H), 0.78 (s, 9H).

Stability of MI-773 (TFA salt): MI-773 (TFA salt) was dissolved in awater/methanol mixture: 1) water/methanol=1:1 with 0.1% of TFA, pH 2.1;2) water/methanol=1:1 with 0.1% of TEA, pH 10.8; or 3)water/methanol=1:1, pH 3.9. The solution was allowed to stand at roomtemperature. The purity was tested using a C18 reverse phase analyticalHPLC column at the time points of 0, 12 h, 24 h, 48 h, and 72 h. Theresults showed transformation of MI-773 (corresponding to peak 3) toMI-77302 (corresponding to peak 1), MI-77301 (corresponding to peak 4)and another compound (corresponding to peak 2) having the same molecularweight (FIGS. 6, 37, and 38). The purity of an identical sample solutionstored at 4° C. was also tested at 0 and 36 h. The results showedcomparably slow transformation of MI-773 at 4° C.

Example 11 Synthesis of MI-77301

MI-773 (as the TFA salt) was dissolved in MeOH/H₂O (1:1 v/v ratio) andallowed to stand at room temperature for 1-4 days. The solution waspurified by chromatography on a C18 reverse phase semi-preparative HPLCcolumn with solvent A (0.1% of TFA in water) and solvent B (0.1% of TFAin methanol) as eluents (gradient: 45% of solvent A and 55% of solvent Bto 30% of solvent A and 70% of solvent B in 30 min). MI-77301 wasisolated as the TFA salt.

¹H NMR (300 MHz, MeOH-d₄): 8.35 (d, J=7.8 Hz, 1H), 7.54-7.62 (m, 2H),7.37-7.43 (m, 1H), 7.12-7.20 (m, 2H), 6.80 (d, J=1.5 Hz, 1H), 5.20 (d,J=11.4 Hz, 1H), 4.58 (d, J=11.4 Hz, 1H), 4.51 (d, J=7.2 Hz, 1H),3.50-3.75 (m, 1H), 3.30-3.50 (m, 1H), 1.82-2.00 (m, 3H), 1.76 (d, J=10.5Hz, 1H), 1.52 (d, J=12.3 Hz, 1H), 1.05-1.42 (m, 4H), 0.88-1.00 (m, 1H),0.88 (s, 9H); ¹³C NMR (75 MHz, MeOH-d₄): 177.7, 166.9, 157.6 (d,JC-F=248.0 Hz), 145.0, 137.0, 132.4, 128.6, 126.6, 126.4 (d, JC-F=4.9),124.0, 123.4, 122.3 (d, JC-F=18.8 Hz), 121.5 (d, JC-F=12.8 Hz), 111.9,69.9, 64.4, 64.0, 62.8, 49.7, 34.3, 34.2, 30.9, 30.82, 30.77, 29.4;ESI-MS calculated for C₂₉H₃₅Cl₂FN₃O₃ (M+H)+ requires 562.20. found562.33; [α]_(D) ²⁵=−27.2° (c=0.005 g/mL in MeOH); Purity (HPLC): >95%(See FIG. 36).

In an alternative procedure, MI-773 (77 mg) was dissolved in 15 mLMeOH/H₂O (v/v=1:1). After 3 days, the needle crystals that had formedwere collected, washed with cold MeOH/H₂O (v/v=1:1) and dried in vacuumto give MI-77301 as the free amine (20 mg; >95% purity as determined byHPLC).

¹H NMR (300 MHz, MeOH-d₄): 7.49-7.55 (m, 1H), 7.25-7.31 (m, 1H),7.10-7.16 (m, 1H), 6.82 (d, J=1.8 Hz, 1H), 6.50-6.71 (m, 1H), 6.49 (d,J=8.4 Hz, 1H), 4.32 (d, J=9.0 Hz, 1H), 4.09 (d, J=8.7 Hz, 1H), 3.57-3.69(m, 1H), 3.49 (d, J=9.2 Hz, 1H), 3.46-3.57 (m, 1H), 1.83-2.07 (m, 3H),1.68-1.80 (m, 1H), 1.54 (dd, J=9.0, 14.3 Hz, 1H), 1.12-1.45 (m, 5H),0.80 (s, 9H). The absolute stereochemical configuration of MI-77301 wasdetermined by X-ray analysis.

Stability of MI-77301 (TFA salt): MI-77301 (TFA salt) was dissolved in awater/methanol mixture (water/methanol=1:1 with 0.1% of TFA). Thesolution was allowed to stand at room temperature. The purity was testedusing a C18 reverse phase analytical HPLC column at the time points of0, 12 h, 48 h, and 72 h. The results showed slow transformation ofMI-77301 (corresponding to peak 4) to MI-77302 (corresponding to peak 1)and two other compounds (corresponding to peaks 2, and 3) having thesame molecular weight (FIG. 7). The absolute stereochemistry of MI-77302was determined by x-ray analysis.

MI-710201, MI-710401, MI-710501, MI-710601, MI-710801, and MI-710901 ofEXAMPLE 1 were prepared using procedures similar to that used to prepareMI-77301.

¹³C CPMAS NMR spectroscopy (400 MHz) of MI-77301 (top), MI-773 (middle),and MI-77302 (bottom) is presented in FIG. 34. Chemical shiftdifferences are observed in the carbonyl region (170-185 ppm).

In an alternative procedure, MI-77301 was prepared as described inScheme 9A.

Example 12 Synthesis of(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide;and(2′R,3S,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide

(5S,6R)-5,6-diphenylmorpholin-2-one was prepared according to J. Org.Chem. 2005, 70, 6653. mp: 139° C. (Kofler); LC-MS: t_(R) (min)=0.96;[M+H]⁺: m/z 254 (method C).

(3R,3′R,4′S,6′S,8′R,8a′S)-6-chloro-8′-(3-chloro-2-fluorophenyl)-6′-neopentyl-3′,4′-diphenyl3′,4′,8′,8a′-tetrahydrospiro[indoline-3,7′-pyrrolo[2,1-c][1,4]oxazine]-1′,2(6′H)-dione

To a suspension of 496 mg (1.61 mmol) of(E)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one in tolueneunder argon, were added 408 mg (1.61 mmol) of(5S,6R)-5,6-diphenylmorpholin-2-one and 213 μL (1.61 mmol) of3,3-dimethyl-butyraldehyde. The reaction mixture was heated at refluxtemperature for 6 hours, upon which it was cooled down to roomtemperature and concentrated to dryness under reduced pressure. Theresidue was purified by flash chromatography on a 70 g silica cartridge(15-40 μm silica gel; eluting solvent: cyclohexane/ethyl acetate 90/10v/v; flow: 50 mL/min). 0.58 g of(3R,3′R,4′S,6′S,8′R,8a′S)-6-chloro-8′-(3-chloro-2-fluorophenyl)-6′-neopentyl-3′,4′-diphenyl3′,4′,8′,8a′-tetrahydrospiro[indoline-3,7′-pyrrolo[2,1-c][1,4]oxazine]-1′,2(6′H)-dionewas obtained as an amorphous yellow solid. LC-MS: t_(R) (min)=1.81;[M+H]⁺: m/z 643[M−H]⁻: m/z 641 (WATERS UPLC-SQD apparatus; Ionization:electrospray in positive mode and/or negative mode (ES+/−);Chromatographic conditions: Column: ACQUITY BEH C18 1.7 μm-2.1×50 mm;Solvents: A: H₂O (0.1% formic acid) B: CH₃CN (0.1% formic acid); Columntemperature: 50° C.; Flow: 0.8 ml/min; Gradient (2.5 min): from 5 to100% of B in 1.8 min; 2.4 min: 100% of B; 2.45 min: 100% of B; from 100to 5% of B in 0.05 min; Retention time=t_(R) (min); referred to hereinas “Method C”); ¹H NMR (400 MHz, DMSO-d₆): 0.39 (s, 9H); 1.30 (dd, J=4.0and 15.2 Hz, 1H); 1.93 (dd, J=6.3 and 15.2 Hz, 1H); 3.49 (dd, J=4.0 and6.3 Hz, 1H); 4.47 (d, J=11.2 Hz, 1H); 5.04 (d, J=4.2 Hz, 1H); 5.08 (d,J=11.2 Hz, 1H); 6.53 (d, J=8.1 Hz, 1H); 6.66 to 6.76 (m, 3H); 6.96 (m,2H); 7.10 to 7.26 (m, 9H); 7.34 (t broad, J=7.9 Hz, 1H); 7.62 (t broad,J=7.9 Hz, 1H); 10.71 (s broad, 1H).

(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-1′-((1S,2R)-2-hydroxy-1,2-diphenylethyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′pyrrolidine]-5′-carboxamide

To a mixture of 112 mg (0.97 mmol) of trans-4-aminocyclohexanol in 2 mLof tetrahydrofuran under argon, was added 0.57 g (0.89 mmol) of(3R,3′R,4′S,6′S,8′R,8a′S)-6-chloro-8′-(3-chloro-2-fluorophenyl)-6′-neopentyl-3′,4′-diphenyl3′,4′,8′,8a′-tetrahydrospiro[indoline-3,7′-pyrrolo[2,1-c][1,4]oxazine]-1′,2(6′H)-dionein 8 mL of tetrahydrofuran. The resulting mixture was heated at 60° C.for 17 hours, upon which 136 μL (0.97 mmol) of triethylamine was addedand heating was pursued.

After 24 hours, 13.6 mg (0.12 mmol) of trans-4-aminocyclohexanol and 2.5mL of tetrahydrofuran were added. After 41 hours, the reaction mixturewas cooled down to room temperature and concentrated to dryness underreduced pressure. The residue was diluted with a mixture of 20 mL ofethyl acetate and 6 mL of water and decanted. The aqueous phase wasextracted with 6 mL of ethyl acetate. The combined organic phases weredried over magnesium sulfate and concentrated to dryness under reducedpressure. The residue was purified by flash chromatography on a 70 gsilica cartridge (15-40 μm silica gel; eluting solvent:cyclohexane/ethyl acetate 50/50 v/v followed by 40/60 v/v; flow: 50mL/min). 0.55 g of(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-1′-((1S,2R)-2-hydroxy-1,2-diphenylethyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′pyrrolidine]-5′-carboxamide was obtained as a white meringue. mp: 190°C. (Kofler); LC-MS: t_(R) (min)=1.56; [M+H]⁺: m/z 758; [M−H]⁻: m/z 756(method C); ¹H NMR (400 MHz, DMSO-d₆): 0.51 (s, 9 H); 0.62 (d, J=15.2Hz, 1H); 0.81 (m, 1H); 1.02 to 1.35 (m, 4H); 1.60 (m, 1H); 1.79 (m, 2H);2.36 (dd, J=9.3 and 15.2 Hz, 1H); 3.24 to 3.34 (m partially hidden, 2H);3.48 (m, 1H); 3.69 (d, J=10.9 Hz, 1H); 3.94 (d, J=7.9 Hz, 1H); 4.13 (d,J=10.9 Hz, 1H); 4.45 (d, J=4.4 Hz, 1H); 5.05 (dd, J=4.4 and 7.9 Hz, 1H); 5.14 (d, J=4.4 Hz, 1H); 6.18 (d, J=8.2 Hz, 1H); 6.50 (d, J=2.0 Hz,1H); 6.59 (t broad, J=7.9 Hz, 1H); 6.67 (dd, J=2.0 and 8.2 Hz, 1H); 6.73(t broad, J=7.9 Hz, 1H); 7.20 (t, J=7.8 Hz, 1H); 7.24 (t broad, J=7.9Hz, 1H); 7.28 (t, J=7.8 Hz, 2H); 7.35 (m, 2H); 7.39 to 7.45 (m, 3H);7.49 (d, J=8.2 Hz, 1H); 7.57 (d, J=7.8 Hz, 2H); 10.32 (s broad, 1H).

(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxy-cyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide

A mixture of 542 mg (0.71 mmol) of(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-1′-((1S,2R)-2-hydroxy-1,2-diphenylethyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamidein 10 mL of ethanol was cooled to 0° C. and 989 mg (1.79 mmol) of cericammonium nitrate was added slowly via spatula in 15 min. The reactionmixture was stirred at 0° C. for 1 hour, upon which it was treated with4 mL of toluene, 2 mL of ethanol, 5 mL of saturated brine and 3 mL ofethyl acetate, and decanted. The organic phase was separated and theaqueous phase was extracted with 2×5 mL of ethyl acetate. The organicphases were combined and washed with 3 mL of 5% sodium carbonate. Afterdecantation, the aqueous phase was diluted with water and reextractedwith 10 mL of ethyl acetate. The organic phases were combined andsuccessively washed with 2 mL of 11% sodium disulfite and 2 mL ofsaturated brine. It was then dried over magnesium sulfate andconcentrated to dryness under reduced pressure. 222 mg of the residuewas purified by flash chromatography on a 40 g silica cartridge (15 μmsilica gel; eluting solvent: dichloromethane/acetone 75/25 v/v followedby 65/35 v/v; flow: mL/min). 0.183 g of an off-white meringue wasobtained, taken up twice in diisopropyl oxide and dried at 25° C. underreduced pressure. 157 mg of(2′S,3R,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxy-cyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamidewere obtained as a white amorphous solid. mp: 176° C. (Kofler); LC-MS:t_(R) (min)=1.13 (91%) and 1.03 (9%); [M+H]⁺: m/z 562; [M−H]⁻: m/z 560(method C); ¹H NMR (400 MHz, DMSO-d₆): 0.76 (s, 9H); 0.85 (dd, J=1.8 and14.3 Hz, 1H); 0.97 to 1.28 (m, 4H); 1.38 to 1.50 (m, 2H); 1.67 (m, 1H);1.80 (m, 2H); 2.56 (t, J=12.1 Hz, 1H); 3.23 to 3.38 (m partially hidden,2H); 3.45 (m, 1H); 3.93 (d, J=9.6 Hz, 1H); 4.22 (dd, J=9.6 and 12.1 Hz,1H); 4.46 (d, J=4.4 Hz, 1 H); 6.62 (d, J=8.1 Hz, 1H); 6.70 to 6.75 (m,2H); 7.13 (t broad, J=7.9 Hz, 1H); 7.31 (t broad, J=7.9 Hz, 1H); 7.64 (tbroad, J=7.9 Hz, 1H); 7.92 (d, J=7.7 Hz, 1 H); 10.51 (s broad, 1H).

(2′R,3S,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxy-cyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide

The rest of the crude compound (206 mg) was dissolved and stirred intomL of ethyl acetate, treated with 41 μL (0.07 mmol) of glacial aceticacid and the reaction mixture was heated at 60° C. for 3 hours, uponwhich it was cooled down to room temperature and stirred for 16 hours.It was then washed with 5 mL of saturated sodium hydrogencarbonate and 3mL of water. The organic phase was dried over magnesium sulfate andconcentrated to dryness under reduced pressure. The residue was purifiedby flash chromatography on a 30 g silica cartridge (15-40 μm silica gel;eluting solvent: dichloromethane/acetone 75/25 v/v; flow: 20 mL/min).The isolated product was taken up twice in diisopropyl oxide. The solidwas filtered and dried at 25° C. under reduced pressure. 106 mg of(2′R,3S,4′R,5′S)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans)-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′pyrrolidine]-5′-carboxamide were obtained as a pinkish amorphous solid.mp: 193° C. (Kofler); LC-MS: t_(R) (min)=1.03; [M+H]⁺: m/z 562; [M−H]⁻:m/z 560 (method C); ¹H NMR (400 MHz, DMSO-d₆): 0.72 (dd, J=1.5 and 14.2Hz, 1H); 0.80 (s, 9H); 1.08 to 1.30 (m, 5H); 1.68 to 1.91 (m, 4H); 3.28to 3.49 (m, 3H); 3.58 (m, 1H); 4.29 (d, J=9.0 Hz, 1H); 4.39 (t, J=9.0Hz, 1H); 4.50 (d, J=4.4 Hz, 1H); 6.68 (d, J=2.0 Hz, 1H); 7.05 (dd, J=2.0and 8.1 Hz, 1H); 7.12 (t broad, J=7.9 Hz, 1H); 7.34 (t broad, J=7.9 Hz,1H); 7.48 to 7.57 (m, 2H); 7.72 (d, J=8.1 Hz, 1H); 10.40 (s broad, 1H);α_(D)=+18.4°+/−0.9 (c=1.525 mg/0.5 mL MeOH).

Example 13 Synthesis of(2′S,3′R,4′S,5′R)-1′-acetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexyl)-amide

Acetic acid4-{(2′S,3′R,4′S,5′R)-1,1′-diacetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carbonyl]-amino}-cyclohexylester

To a solution of 281 mg (0.50 mmol) of(2′S,3′R,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylic acid(trans-4-hydroxy-cyclohexyl)-amide in 5.0 mL of pyridine under argon,was added 178 μL (2.50 mmol) of acetyl chloride. The resulting mixturewas stirred at room temperature for 4 days, upon which it was pouredinto a mixture of water and ethyl acetate. The organic phase wasseparated and the aqueous phase was extracted twice with ethyl acetate.The combined organic extracts were washed with brine, dried withmagnesium sulfate and then concentrated to dryness under reducedpressure. The residue was purified by flash chromatography on a 50 gsilica cartridge (15-40 μm silica gel; eluting solvent:dichloromethane/methanol 98/2 v/v; flow: 40 mL/min) followed by a secondpurification by flash chromatography on a 30 g silica cartridge (15-40μm silica gel; eluting solvent: dichloromethane/methanol 98/2 v/v; flow:mL/min). 123 mg of acetic acid4-{[(2′S,3′R,4′S,5′R)-1,1′-diacetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidin]e-5′-carbonyl]-amino}-cyclohexyl ester wereobtained as a white powder. LC-MS: t_(R) (min)=1.19; [M+H]⁺: m/z 688;[M−H]⁻: m/z 686 (method A).

(2′S,3′R,4′S,5′R)-1′-Acetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexyl)-amide

To a solution of 117 mg (0.17 mmol) of acetic acid4-{[(2′S,3′R,4′S,5′R)-1,1′-diacetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carbonyl]-amino}-cyclohexylester in 10.0 mL of methanol under argon, was added 10 mL (81 mmol) of asaturated potassium carbonate solution. The resulting mixture wasstirred at room temperature for 1 hour, upon which the methanol wasevaporated under reduced pressure. The remaining aqueous phase wasextracted 3 times with 20 mL of dichloromethane. The combined organicextracts were washed with 20 mL of brine, dried with magnesium sulfateand then concentrated to dryness under reduced pressure. 92 mg of(2′S,3′R,4′S,5′R)-1′-acetyl-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexyl)-amide were obtained as a white powder.mp: 220° C. (Kofler); LC-MS: t_(R) (min)=0.97; [M+H]⁺: m/z 604; [M−H]⁻:m/z 602 (method A); ¹H NMR (60° C., CHLOROFORM-d, 400 MHz): 0.68 (s,9H); 0.75 to 2.62 (m partially hidden, 14 H); 3.51 (m, 1H); 3.65 (m,1H); 4.28 (m broad, 1H); 4.48 (dd, J=3.4 and 5.9 Hz, 1H); 4.98 (d,J=10.3 Hz, 1H); 6.65 (d, J=1.5 Hz, 1H); 6.99 (t, J=7.8 Hz, 1H); 7.05 to7.48 (m, 5H).

Example 14 Synthesis of(2′S,3′R,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide

(2′R,3′S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-1′-((1R,2S)-2-hydroxy-1,2-diphenyl-ethyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide

To a solution of 0.28 g (1.71 mmol) of 4-aminomethyl-cyclohexanolhydrochloride in 12.0 mL of tetrahydrofuran, was added 0.46 mL (3.26mmol) of triethylamine. The resulting mixture was stirred at roomtemperature for 30 minutes and 1 g (1.55 mmol) of:

was added progressively via spatula, followed by 2 mL oftetrahydrofuran. The reaction mixture was heated at reflux temperaturefor 7 hours and then stirred at room temperature for 2.5 days, uponwhich it was diluted with 10 mL of water and 15 mL of ethyl acetate. Theorganic phase was separated and the aqueous phase was extracted withethyl acetate. The combined organic extracts were dried with magnesiumsulfate and then concentrated to dryness under reduced pressure. Theresidue was purified by flash chromatography on a 70 g silica cartridge(15-40 μm silica gel; eluting solvent: dichloromethane, thendichloromethane/methanol 98/2 v/v; flow: 50 mL/min). 0.40 g of(2′R,3′S,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-1′-((1R,2S)-2-hydroxy-1,2-diphenyl-ethyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide were obtained as a whitesolid.

LC-MS: t_(R) (min)=1.16; [M+H]⁺: m/z 772; [M−H]⁻: m/z 770 (method A); ¹HNMR (CHLOROFORM-d, 400 MHz): 0.74 (s large, 9H); 0.83 (m, 2H); 1.13 (m,2H); 1.20 (m, 1H); 1.25 (d, J=15.6 Hz, 1H); 1.35 (d, J=4.9 Hz, 1H); 1.42(m, 1H); 1.55 (m partially hidden, 1H); 1.84 to 2.00 (m, 2H); 2.29 (d,J=2.9 Hz, 1H); 2.67 (dd, J=9.3 and 15.6 Hz, 1H); 2.86 (m, 1H); 3.38 (m,1H); 3.48 (m, 2 H); 4.11 (m, 2H); 4.36 (d, J=8.3 Hz, 1H); 5.20 (dd,J=2.9 and 8.3 Hz, 1H); 5.64 (d, J=8.1 Hz, 1H); 6.28 (m, 1H); 6.44 (t,J=7.8 Hz, 1H); 6.53 (d, J=2.2 Hz, 1 H); 6.62 (dd, J=2.2 and 8.1 Hz, 1H);6.67 (t, J=7.8 Hz, 1H); 7.09 (t, J=7.8 Hz, 1 H); 7.25 (s, 1H); 7.30 to7.45 (m, 5H); 7.54 to 7.67 (m, 5H).

(2′S,3′R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide

In a three-neck 50 mL flask were successively introduced 0.70 g (0.91mmol) of(2′R,3′S,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-1′-((1R,2S)-2-hydroxy-1,2-diphenyl-ethyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide, 7.0 mL of acetonitrile,3.5 mL of distilled water and 3.5 mL of acetone. The resulting mixturewas stirred and cooled to 0° C. and 0.99 g (1.81 mmol) of ceriumammonium nitrate was added in small portions. The reaction mixture wasstirred at 0° C. for 20 minutes, upon which 89 mg (1.06 mmol) of sodiumhydrogencarbonate were added and stirring was maintained for minutes.The mixture was diluted with 60 mL of ethyl acetate and decanted. Theorganic phase was separated and the aqueous phase was extracted twicewith mL of ethyl acetate. The combined organic extracts were washed with20 mL of water, dried with magnesium sulfate and then concentrated todryness under reduced pressure. The residue was purified by flashchromatography on a 30 g silica cartridge (15-40 μm silica gel; elutingsolvent: dichloromethane; flow: 30 mL/min) followed by a secondpurification by flash chromatography on a 15 g silica cartridge (15-40μm silica gel; eluting solvent: dichloromethane/methanol/28% ammonia97/2/1 v/v/v; flow: 30 mL/min). 87 mg of(2′S,3′R,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexylmethyl)-amide were obtained as a whitesolid. mp: 192° C. (Kofler); LC-MS: t_(R) (min)=0.84; [M+H]⁺: m/z 576;[M−H]⁻: m/z 574 (method A); ¹H NMR (CHLOROFORM-d, 400 MHz): mixture ofisomers: 0.90 (s, 9H); 0.99 to 2.09 (m partially hidden, 11H); 3.05 to3.27 (m, 2H); 3.59 (m, 2H); 4.39 (d, J=8.8 Hz, 1H); 4.59 (m, 1H); 6.76(s large, 1H); 6.99 (t, J=7.8 Hz, 1H); 7.06 to 7.56 (m, 6H); 7.82 (tbroad, J=6.1 Hz, 1H).

Example 15 Synthesis of(2′S,3′R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-1′-methyl-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (trans-4-hydroxy-cyclohexyl)-amide

To a suspension of 0.50 g (0.89 mmol) of(2′S,3′R,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylic acid(trans-4-hydroxy-cyclohexyl)-amide in 18.0 mL of acetonitrile underargon, were added 0.89 mL (0.97 mmol) of a 36.5% solution offormaldehyde in water, followed by 65 mg (0.98 mmol) of sodiumcyanoborohydride. The resulting solution was stirred at room temperaturefor 2 hours, upon which it was poured into 50 mL of ethyl acetate. Theaqueous phase was separated and the organic phase was washed with asaturated solution of sodium hydrogencarbonate. The latter aqueous phasewas reextracted with ethyl acetate. The combined organic extracts werewashed with brine, dried with magnesium sulfate and then concentrated todryness under reduced pressure. The residue (0.52 g) was purified bychiral HPLC chromatography on a Kromasil C18 column (1100 g batch 4680,10 m, 7.65×35 cm), eluting solvent: acetonitrile/water 40/60 v/v+0.1%trifluoroacetic acid; flow: 250 mL/min. The collected solution wastreated with sodium hydrogencarbonate up to pH 8 and then extracted 3times with 200 mL of ethyl acetate. The combined organic extracts werewashed twice with 100 mL of water, dried with magnesium sulfate and thenconcentrated to dryness under reduced pressure. The residue was thendried in a dessicator under reduced pressure for 16 hours. 0.17 g of(2′S,3′R,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluoro-phenyl)-2′-(2,2-dimethyl-propyl)-1′-methyl-2-oxo-1,2-dihydro-spiro[indole-3,3′-pyrrolidine]-5′-carboxylicacid (4-hydroxy-cyclohexyl)-amide was obtained as a white solid. mp:188° C. (Kofler); LC-MS:

t_(R) (min)=0.85-0.97 (mixture of isomers); [M+H]⁺: m/z 576; [M−H]⁻: m/z574 (method A); ¹H NMR (400 MHz, CHLOROFORM-d): 0.73 (d, J=15.6 Hz, 1H);0.79 (s, 9H); 1.15 to 1.48 (m, 4H); 1.85 to 2.12 (m, 5H); 2.75 (s, 3H);3.60 to 3.77 (m, 3H); 4.17 (d, J=9.8 Hz, 1H); 4.33 (d broad, J=9.8 Hz,1H); 6.69 (d, J=1.5 Hz, 1H); 6.99 (t, J=7.8 Hz, 1H); 7.05 (dd, J=1.5 and8.3 Hz, 1H); 7.10 to 7.25 (m, 3H); 7.37 (m broad, 1H); 7.56 (t, J=7.8Hz, 1H).

Similar methodology was used to prepare C29701 and C30201.

Example 16 Isomerization Studies General Information

Experiments involving moisture and/or air sensitive components wereperformed in oven-dried glassware under an atmosphere of nitrogen.Commercial solvents and reagents were used without further purificationwith the following exception: THF was freshly distilled from sodiumwire.

Flash chromatography was performed using silica gel (type H) from TMchemicals, Inc. Columns were typically packed as slurry and equilibratedwith hexane prior to use. Analytical thin layer chromatography (TLC) wasperformed using Merck 60 F254 precoated silica gel plate (0.2 mmthickness). Subjected to elution, plates were visualized using UVradiation. Further visualization was possible by staining with basicsolution of potassium permanganate or acidic solution of phosphomolybdicacid, followed by heating with heating gun.

Compounds were purified by HPLC using a Waters Sunfire C18 reverse phasesemipreparative HPLC column (19 mm×150 mm) using solvent A (water, 0.1%of TFA) and solvent B (CH₃CN, 0.1% of TFA or MeOH, 0.1% of TFA) aseluents with a flow rate of 10 mL/min on a Waters Delta 600 instrument.Analytical reverse phase HPLC was conducted using Waters 2795 Separationmodule.

Proton nuclear magnetic resonance (¹H NMR) and carbon nuclear magneticresonance (¹³C NMR) spectroscopy were performed on a Bruker Advance 300NMR spectrometer. Chemical shifts of ¹H NMR spectra are reported as δ inunits of parts per million (ppm) downfield from SiMe₄ (δ 0.0) orrelative to the signal of chloroform-d (δ=7.26, singlet), methanol-d₄(δ=3.31, quintuplet) and DMSO-d₆ (δ=2.50, quintuplet). Multiplicitieswere given as: s (singlet); d (doublet); t (triplet); q (quartet); dd(doublet of doublets); ddd (doublet of doublets of doublets); dt(doublet of triplets); m (multiplets) and etc. The number of protons fora given resonance is indicated by nH. Coupling constants are reported asJ values in Hz. Carbon nuclear magnetic resonance spectra (¹³C NMR) arereported as δ in units of parts per million (ppm) downfield from SiMe₄(δ 0.0) or relative to the signal of chloroform-d (δ=77.23, triplet),MeOH-d₄ (δ=49.20, septuplet) and DMSO-d₆ (δ=39.52, septuplet).

Low resolution ESI mass spectrum analysis was performed onThermo-Scientific LCQ Fleet mass spectrometer.

Compounds 1-10 were prepared according to Scheme 10 and Table 4 usingmethods previous described (See, e.g., Ding, K. et al., J. Am. Chem.Soc. 127:10130-10131 (2005); Ding, K. et al., J. Med. Chem. 49:3432-3435(2006); Yu, S. et al., J. Med. Chem. 52:7970-7973 (2009); Shangary, S.,Proc. Natl. Acad. Sci. 105:3933-3938 (2008); U.S. Pat. No. 7,759,383) asa mixture of isomers. Isomer A was identified as the predominant isomerfollowing CAN oxidation in most cases. Applicants have found thatdissolving the mixture of isomers obtained from CAN oxidation in asolvent or a mixture of solvents and allowing the reaction mixture tomature for a period of time under various conditions provides a mixtureof isomers having Isomer B as the predominant isomer. In some cases,Isomers C and D were isolated in pure or substantially pure form.Likewise, compounds 11 and 12 were prepared according to Scheme 11 andTable 5.

The procedure for isomerization used in this study was as follows:approximately 30 mg product obtained from CAN oxidation (pre-purified byflash column chromatography) was placed in a round bottom flask equippedwith magnetic stirring bar. Acetonitrile (2.4 mL) was added to dissolvethe product. To the acetonitrile solution was added water (2.0 mL) and0.5 mL NaHCO₃ (saturated) solution to give a pH of approximately 8. Thereaction mixture was allowed to stir at room temperature forapproximately 3 days. The percentage of isomers was determined usinganalytical HPLC. Further purification was performed on semi-preparativeor preparative reverse phase HPLC using MeOH (0.1% TFA) and water (0.1%TFA) as mobile phase.

Isomerization of Isomer A to Isomer B can also be carried out underacidic conditions, e.g., MeCN—H₂O, CF₃CO₂H (pH<1), room temperature, 3days; ethyl acetate, acetic acid, 60° C., 3 h, or neutral conditionse.g., MeOH or MeOH—H₂O.

TABLE 4 Isomer Isomer Ratio Ratio after after Yield^(a) OxidationIsomerization Cpd R₁ R₂ —NR^(b)R^(c) (%) A:B:(C + D)^(b) A:B:(C + D)^(b) 1 H H —NMe₂  50^(c) 95:2:3 6:71:24  2 H H —NHMe 61 87:5:8 3:71:26  3 HF —NMe₂ 57 75:19:6 35:46:19  4 H F NHMe 84 67:2:31 9:79:12  5 H F

86 88:2:10 1:80:19  6 H F

79 89:10:1 12:61:27  7 H F —NH₂ 56 0:44:56 0:96:4  8 F H —NMe₂ 6056:25:19 10:71:19^(d) 2:88:8  9 F H —NHMe 87 19:52:27 1:74:25 10^(e) F H

81 30:32:38 3:58:37 ^(a)Yield after oxidation; ^(b)Ratio was determinedby HPLC analysis; ^(c)Yield after HPLC separation; ^(d)Ratio determinedby HPLC analysis after allowing oxidation product to stand in MeOH fortwo hours; ^(e)Compound 10 Isomer A and Compound 10 Isomer B arereferred to as MI-219 and MI-21901, respectively, in Table 2A.

TABLE 5 Isomer Isomer Ratio after Ratio after Yield OxidationEquilibration Cpd (%) a:b:(c + d)^(b) a:b:(c + d)^(b) 11 74^(a) 89:6:344:50:6 12 25^(c) — 41:34:25^(d) ^(a)Yield after oxidation; ^(b)Ratiowas determined by HPLC analysis; ^(c)Hydrolysis yield; ^(d)Starting withpure Compound 12-Isomer A.

Analytical Data

Compound 1—Isomer A (TFA salt): ESI: Calculated for C₂₅H₃₀ ³⁵Cl₂N₃O₂[M+H]⁺=474.17. Found: 474.50.

Compound 1—Isomer B (TFA salt): ESI: Calculated for C₂₅H₃₀ ³⁵Cl₂N₃O₂[M+H]⁺=474.17. Found: 474.68.

Compound 1—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.72 (d, J=8.08Hz, 1H), 7.28-7.22 (m, 1H), 7.20-7.12 (m, 2H), 7.12-7.08 (m, 1H), 7.04(d, J=7.65 Hz, 1H), 6.81 (d, J=1.79 Hz, 1H), 5.54 (d, J=10.87 Hz, 1H),4.37 (dd, J=6.54, 4.52 Hz, 1H), 4.15 (d, J=10.85 Hz, 1H), 2.98 (s, 3H),2.81 (s, 3H), 1.70 (dd, J=15.24, 6.67 Hz, 1H), 1.20 (dd, J=15.26, 4.43Hz, 1H), 0.91 (s, 9H); ESI: Calculated for C₂₅H₃₀ ³⁵Cl₂N₃O₂[M+H]⁺=474.17. Found: 474.50.

Compound 2—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.28-7.16 (m,3H), 7.16-7.06 (m, 1H), 6.92-6.82 (m, 2H), 6.80-6.76 (m, 1H), 4.92 (d,J=10.23, 4.20-4.10 (m, 2H), 2.73 (s, 3H), 1.99 (s, J=15.32, 6.75 Hz,1H), 1.47 (dd, J=15.54, 3.49 Hz, 1H), 0.80 (s, 9H); ESI: Calculated forC₂₄H₂₈ ³⁵Cl₂N₃O₂ [M+H]⁺=460.16. Found: 460.52.

Compound 2—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.63 (d, J=8.06Hz, 1H), 7.30-7.14 (m, 4H), 7.12-7.00 (m, 1H), 6.82-6.76 (m, 1H), 5.29(d, J=11.24 Hz, 1H), 4.47 (d, J=6.68 Hz, 1H), 4.16 (d, J=11.22 Hz, 1H),2.73 (s, 3H), 1.92 (dd, J=15.40, 8.39 Hz, 1H), 1.17 (d, J=16.85 Hz, 1H),0.90 (s, 9H); ¹³C (75 MHz, CD₃OD): 177.78, 168.54, 145.38, 137.07,135.77, 134.60, 131.40, 130.29, 129.48, 128.29, 126.32, 124.24, 124.16,112.18, 65.11, 64.18, 62.85, 56.95, 43.42, 30.97, 29.66, 26.98; ESI:Calculated for C₂₄H₂₈ ³⁵Cl₂N₃O₂ [M+H]⁺=460.16. Found: 460.48.

Compound 2—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.57 (d, J=8.12Hz, 1H), 7.24-7.08 (m, 3H), 7.04-6.98 (m, 1H), 6.92 (d, J=7.62 Hz, 1H),6.76 (d, J=1.78 Hz, 1H), 5.02 (d, J=12.47 Hz, 1H), 4.42 (dd, J=7.06,4.40 Hz, 1H), 4.13 (d, J=12.47 Hz, 1H), 2.70 (s, 3H), 1.74 (dd, J=15.35,7.14 Hz, 1H), 1.17 (dd, J=15.37, 4.38 Hz, 1H), 0.88 (s, 9H); ¹³C (75MHz, CD₃OD): 176.72, 167.32, 144.88, 137.13, 135.62, 133.98, 131.17,130.24, 129.73, 128.41, 127.88, 123.91, 123.53, 112.57, 65.42, 64.33,62.97, 59.03, 44.86, 31.01, 29.57, 27.03; ESI: Calculated for C₂₄H₂₈³⁵Cl₂N₃O₂ [M+H]⁺=460.16. Found: 460.50.

Compound 3—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.80-7.70 (m,1H), 7.50-7.40 (m, 1H), 7.32-7.22 (m, 1H), 6.92-6.88 (m, 1H), 6.74 (dd,J=8.15, 1.75 Hz, 1H), 6.48 (d, J=8.11 Hz, 1H), 5.58 (d, J=7.94 Hz, 1H),4.38 (d, J=7.94 Hz, 1H), 4.26 (d, J=7.55 Hz, 1H), 2.99 (s, 3H), 2.84 (s,3H), 2.06 (dd, J=15.42, 7.72 Hz, 1H), 1.13 (d, J=15.37 Hz, 1H), 0.89 (s,9H); ¹³C (75 MHz, CD₃OD): 180.07, 166.93, 157.51 (d, J_(C-F)=243.98 Hz),145.59, 137.11, 132.54, 128.77, 127.66, 126.74 (d, J_(C-F)=4.55 Hz),125.89 (d, J_(C-F)=13.40 Hz), 123.68, 122.85 (d, J_(C-F)=10.19 Hz),123.10 (d, J_(C-F)=18.37 Hz), 112.07, 63.03, 62.05, 61.76, 42.09, 37.70,36.89, 30.77, 29.34; ESI: Calculated for C₂₅H₂₉ ³⁵Cl₂FN₃O₂[M+H]⁺=492.16. Found: 492.44.

Compound 3—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.68-7.57 (m,2H), 7.45-7.35 (m, 1H), 7.22-7.10 (m, 2H), 6.84-6.77 (m 1H), 5.68 (d,J=10.24 Hz, 1H), 4.64 (d, J=10.24 Hz, 1H), 4.48 (dd, J=8.20, 1.70 Hz,1H), 2.98 (s, 3H), 2.80 (s, 3H), 1.89 (dd, J=15.48, 8.26 Hz, 1H), 1.14(dd, J=15.48, 1.67 Hz, 1H), 0.89 (s, 9H); ¹³C (75 MHz, CD₃OD): 177.96,168.37, 152.99 (d, J_(C-F)=253.32 Hz), 145.26, 137.39, 132.82, 128.82,126.95, 126.70 (d, J_(C-F)=4.79 Hz), 124.28, 122.09 (d, J_(C-F)=13.13Hz), 118.90, 115.11, 112.24, 64.69, 59.97, 42.64, 37.96, 37.03, 31.03,29.62; ESI: Calculated for C₂₅H₂₉ ³⁵Cl₂FN₃O₂ [M+H]⁺=492.16. Found:492.46.

Compound 3—Isomer C (TFA salt): ¹H NMR (300 MHz, CD₃OD) (TFA salt): 7.63(d, J=8.13 Hz, 1H), 7.52-7.38 (m, 2H), 7.24-7.12 (m, 2H), 6.83 (d,J=1.80 Hz, 1H), 5.47 (d, J=11.16 Hz, 1H), 4.42 (d, J=11.31 Hz, 1H), 4.35(t, J=5.66 Hz, 1H), 2.95 (s, 3H), 2.93 (s, 3H), 1.91 (dd, J=15.39, 5.80Hz, 1H), 1.71 (dd, J=15.24, 5.43 Hz, 1H), 0.84 (s, 9H); ESI: Calculatedfor C₂₅H₂₉ ³⁵Cl₂FN₃O₂ [M+H]⁺=492.16. Found: 494.20.

Compound 3—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD) (TFA salt): 7.60(d, J=8.07 Hz, 1H), 7.40-7.30 (m, 1H), 7.22-7.12 (m, 1H), 7.07 (dd,J=8.16, 1.87 Hz, 1H), 7.04-6.95 (m, 1H), 6.80 (d, J=1.87 Hz, 1H), 5.58(d, J=9.80 Hz, 1H), 4.51 (d, J=9.84 Hz, 1H), 4.43 (dd, J=6.69, 4.15 Hz,1H), 2.99 (s, 3H), 2.80 (s, 3H), 1.68 (dd, J=15.41, 6.81 Hz, 1H), 1.42(dd, J=15.41, 4.26 Hz, 1H), 0.90 (s, 9H); ESI: Calculated for C₂₅H₂₉³⁵Cl₂FN₃O₂ [M+H]⁺=492.16. Found: 492.28.

Compound 4—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.66-7.54 (m,1H), 7.38-7.28 (m, 1H), 7.22-7.10 (m, 1H), 6.87 (s, 1H), 6.82-6.72 (m,2H), 5.21 (d, J=10.00 Hz, 1H), 4.50 (d, J=9.93 Hz, 1H), 4.30-4.24 (m,1H), 2.75 (s, 3H), 2.07 (dd, J=15.35, 7.35 Hz, 1H), 1.8 (d, J=14.57 Hz,1H), 0.80 (s, 9H); ¹³C (75 MHz, CD₃OD): 180.31, 167.24, 157.81 (d,J_(C-F)=247.78 Hz), 145.44, 136.94, 132.11, 128.66, 127.94, 126.47 (d,J_(C-F)=4.45 Hz), 125.29, 125.01 (d, J_(C-F)=13.96 Hz), 123.37, 122.64(d, J_(C-F)=18.03 Hz), 122.04, 64.35, 63.69, 61.74, 49.51, 42.34, 30.92,29.55, 27.10; ESI: Calculated for C₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15.Found: 478.92.

Compound 4—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.65-7.55 (m,2H), 7.40-7.34 (m, 1H), 7.20-7.10 (m, 2H), 6.84-6.80 (m, 1H), 5.26 (d,J=11.15 Hz, 1H), 4.64 (d, J=11.19 Hz, 1H), 4.45 (d, J=7.86 Hz, 1H), 2.74(s, 3H), 1.87 (dd, J=15.19, 8.58 Hz, 1H), 1.11 (d, J=15.21 Hz, 1H), 0.90(s, 9H); 13C (75 MHz, CD₃OD): 177.75, 168.20, 159.53 (d, J_(C-F)=248.25Hz), 145.22, 137.24, 132.59, 128.58, 126.63 (d, J_(C-F)=5.25 Hz),124.16, 123.43, 126.66, 122.59 (d, J_(C-F)=4.05 Hz), 121.54 (d,J_(C-F)=12.75 Hz), 112.13, 64.49, 64.40, 62.73, 55.34, 43.33, 31.01,29.58, 27.06; ESI: Calculated for C₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15.Found: 478.25.

Compound 4—Isomer C (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.62 (d, J=8.17Hz, 1H), 7.46-7.36 (m, 2H), 7.22 (dd, J=8.13, 1.89 Hz, 1H), 7.15 (t,J=8.08 Hz, 1H), 6.80 (d, J=1.85 Hz, 1H), 4.84 (d, J=12.32, 1H), 4.35 (d,J=12.32 Hz, 1H), 4.24 (t, J=5.62 Hz, 1H), 2.67 (s, 3H), 1.87 (dd,J=5.44, 2.53 Hz, 2H), 0.77 (s, 9H); ESI: Calculated for C₂₄H₂₇³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.52.

Compound 4—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.53 (d, J=8.11Hz, 1H), 7.36-7.26 (m, 1H), 7.11 (dd, J=8.08, 1.88 Hz, 1H), 6.98-6.90(m, 2H), 6.77 (d, J=1.85 Hz, 1H), 5.03 (d, J=11.95 Hz, 1H), 4.51 (d,J=11.87 Hz, 1H), 4.50-4.42 (m, 1H), 2.72 (s, 3H), 1.72 (d, J=15.36, 7.30Hz, 1H), 1.16 (dd, J=15.37, 4.09 Hz, 1H), 0.89 (s, 9H); ESI: Calculatedfor C₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.38.

Compound 5—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.63-7.53 (m,1H), 7.40-7.30 (m, 1H), 7.23-7.13 (m, 1H), 6.87 (d, J=1.36 Hz, 1H),6.86-6.74 (m, 2H), 5.19 (d, J=10.22 Hz, 1H), 4.49 (d, J=10.22 Hz, 1H),4.25 (dd, J=7.35, 2.71 Hz, 1H), 3.56-3.43 (m, 1H), 3.42-3.30 (m, 4H),2.07 (dd, J=15.43, 7.44 Hz, 1H), 1.72-1.58 (m, 1H), 1.56-1.40 (m, 1H),1.30 (dd, J=15.43, 2.62 Hz, 1H), 0.80 (s, 9H); ¹³C (75 MHz, CD₃OD):180.26, 166.78, 157.81 (d, J_(C-F)=247.93 Hz), 145.43, 136.90, 132.16,128.82, 127.96, 126.48 (d, J_(C-F)=4.49 Hz), 125.43, 124.94 (d,J_(C-F)=14.00 Hz), 123.37, 122.63 (d, J_(C-F)=18.23 Hz), 112.00, 71.01,67.25, 64.38, 63.80, 61.69, 50.06, 42.36, 38.30, 33.80, 30.94, 29.55;ESI: Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18. Found: 552.92.

Compound 5—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.60 (d, J=8.15Hz, 1H), 7.55 (t, J=7.12 Hz, 1H), 7.38 (t, J=7.57 Hz, 1H), 7.20-7.10 (m,2H), 6.78 (d, J=1.70 Hz, 1H), 5.27 (d, J=11.40 Hz, 1H), 4.62 (d, J=11.40Hz, 1H), 4.52 (dd, J=8.33, 1.29 Hz, 1H), 3.45-3.25 (m, 5H), 1.90 (dd,J=15.46, 8.38 Hz, 1H), 1.64-1.48 (m, 1H), 1.46-1.32 (m, 1H), 1.14 (dd,J=15.46, 1.34 Hz, 1H), 0.87 (s, 9H); ¹³C (75 MHz, CD₃OD): 177.77,167.67, 157.90 (d, J_(C-F)=251.2 Hz), 145.21, 137.26, 132.67, 128.64,126.89 (d, J_(C-F)=1.88 Hz), 126.65 (d, J_(C-F)=4.89 Hz), 124.17,123.43, 122.60 (d, J_(C-F)=19.0 Hz), 121.52 (d, J_(C-F)=13.0 Hz),112.14, 79.99, 67.25, 64.45, 62.873, 48.8, 43.39, 38.26, 33.78, 31.02,29.57; ESI: Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18. Found:552.42;

Compound 5—Isomer C (TFA salt): ¹H NMR (300 MHz, CD₃OD): 8.37 (s, broad,NH), 7.62 (d, J=8.11 Hz, 1H), 7.50-7.34 (m, 2H), 7.36-7.10 (m, 2H), 6.83(d, J=1.83 Hz, 1H), 4.32 (d, J=12.09 Hz, 1H), 4.24-4.14 (m, 1H),3.50-3.10 (m, 5H), 1.86-1.72 (m, 2H), 1.58-1.44 (m, 1H), 1.44-1.26 (m,1H), 0.80 (s, 9H); ESI: Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18.Found: 552.42.

Compound 5—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.49 (d, J=8.02Hz, 1H), 7.36-7.24 (m, 1H), 7.11 (dd, J=8.09, 1.89 Hz, 1H), 7.06-6.96(m, 1H), 6.96-6.88 (m, 1H), 6.80 (d, J=1.88 Hz, 1H), 4.89 (d, J=11.57Hz, 1H), 4.46 (d, J=11.57 Hz, 1H), 4.35-4.25 (m, 1H), 3.56-3.40 (m, 1H),3.40-3.20 (m, 4H), 1.70-1.40 (m, 3H), 1.10 (dd, J=14.59, 3.06 Hz, 1H),0.91 (s, 9H); ESI: Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18.Found: 552.40.

Compound 6—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.59 (t, J=6.93Hz, 1H), 7.41 (td, J=7.55, 0.97 Hz, 1H), 7.22 (t, J=7.90 Hz, 1H), 6.90(d, J=1.79 Hz, 1H), 6.77 (dd, J=8.14, 1.79 Hz, 1H), 6.48 (d, J=8.15 Hz,1H), 5.21 (d, J=8.22 Hz, 1H), 4.50 (d, J=8.19 Hz, 1H), 4.17 (dd, J=7.63,2.23 Hz, 1H), 4.10-3.80 (m, 4H), 3.75-3.45 (m, 4H), 3.40-3.00 (m, 4H),2.08-1.96 (m, 1H), 1.19 (dd, J=15.32, 2.23 Hz, 1H), 0.81 (s, 9H); ¹³C(75 MHz, CD₃OD): 180.28, 168.91, 157.75 (d, J_(C-F)=248.38 Hz), 145.50,137.13, 132.29, 128.63, 127.67, 126.63 (d, J_(C-F)=4.50 Hz), 125.74 (d,J_(C-F)=13.69 Hz), 124.72, 123.44, 122.83 (d, J_(C-F)=18.25 Hz), 112.20,65.16, 64.25, 63.84, 62.22, 58.01, 53.84, 50.05, 42.39, 35.72, 30.95,29.52; ESI: Calculated for C₂₉H₃₆ ³⁵Cl₂FN₄O₃ [M+H]⁺=577.21. Found:577.48.

Compound 6—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.66-7.48 (m,2H), 7.42-7.32 (m, 1H), 7.20-7.10 (m, 2H), 6.78 (d, J=1.68 Hz, 1H), 5.38(d, J=11.48 Hz, 1H), 4.65 (d, J=11.48 Hz, 1H), 4.52 (d, J=7.59 Hz, 1H),4.00-3.70 (m, 4H), 3.70-3.60 (m, 2H), 3.50-3.40 (m, 2H), 3.40-3.10 (m,4H), 1.97 (dd, J=15.40, 8.62 Hz, 1H), 1.12 (d, J=15.40 Hz, 1H), 0.88 (s,9H); 13C (75 MHz, CD₃OD): 177.73, 169.20, 145.19, 137.15, 132.54,128.69, 126.84, 126.64 (d, J_(C-F)=4.80 Hz), 124.11, 123.72, 122.57 (d,J_(C-F)=13.19 Hz), 121.77 (d, J_(C)-F=13.19 Hz), 112.06, 65.08, 64.59,64.32, 62.69, 57.41, 53.70, 48.47, 43.55, 35.61, 31.06, 29.56; ESI:Calculated for C₂₉H₃₆ ³⁵Cl₂FN₄O₃ [M+H]⁺=577.21. Found: 577.48.

Compound 7—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.54-7.44 (m,2H), 7.36-7.26 (m, 1H), 7.14-7.00 (m, 2H), 6.70 (d, J=1.76 Hz, 1H), 5.22(d, J=11.36 Hz, 1H), 4.50 (d, J=11.36 Hz, 1H), 4.41 (d, J=8.23, 1.85 Hz,1H), 1.81 (dd, J=15.46, 8.31 Hz, 1H), 1.06 (dd, J=15.46, 1.90 Hz, 1H),0.78 (s, 9H); ESI: Calculated for C₂₃H₂₅ ³⁵Cl₂FN₃O₂ [M+H]⁺=464.13.Found: 464.60.

Compound 7—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.64 (d, J=8.01Hz, 1H), 7.54-7.40 (m, 2H), 7.28-7.12 (m, 2H), 6.83 (d, J=1.68 Hz, 1H),4.96 (d, J=12.33 Hz, 1H), 4.36 (d, J=12.33 Hz, 1H), 4.28 (t, J=5.60 Hz,1H), 1.93-1.86 (m, 2H), 0.80 (s, 9H); ESI: Calculated for C₂₃H₂₅³⁵Cl₂FN₃O₂ [M+H]⁺=464.13. Found: 464.42.

Compound 8—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.66 (d, J=8.49HZ, 1H), 7.35-7.12 (m, 4H), 6.86 (d, J=6.02 Hz, 1H), 5.67 (d, J=10.03Hz, 1H), 4.41 (dd, J=8.20, 1.79 Hz, 1H), 4.13 (d, J=10.03 Hz, 1H), 2.97(s, 3H), 2.74 (s, 3H), 1.91 (dd, J=15.46, 8.20 Hz, 1H) 1.17 (dd,J=15.46, 1.82 Hz, 1H), 0.91 (s, 9H); ¹³C (75 MHz, CD₃OD):177.80, 168.54,156.04 (d, J_(C-F)=243.74 Hz), 141.04, 136.12, 135.16, 131.81, 130.66,129.73, 128.52, 125.50 (d, J_(C-F)=7.37 Hz), 123.70 (d, J_(C-F)=19.58Hz), 114.43 (d, J_(C-F)=25.50 Hz), 113.62, 65.81, 64.45, 60.58, 57.31,42.59, 38.04, 37.00, 31.04, 29.63; ESI: Calculated for C₂₅H₂₉ ³⁵Cl₂FN₃O₂[M+H]⁺=492.16. Found: 492.50.

Compound 8—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.73 (d, J=8.75Hz, 1H), 7.30-7.18 (m, 1H), 7.18-7.12 (m, 1H), 7.12-7.06 (m, 1H), 6.89(d, J=6.12 Hz, 1H), 5.52 (d, J=10.56 Hz, 1H), 4.37 (dd, J=6.56, 4.48 Hz,1H), 4.18 (d, J=10.62 Hz, 1H), 2.99 (s, 3H), 2.80 (s, 3H), 1.69 (dd,J=15.35, 6.58 Hz, 1H), 1.21 (dd, J=15.35, 4.37 Hz, 1H), 0.93 (s, 9H);ESI: Calculated for C₂₅H₂₉ ³⁵Cl₂FN₃O₂ [M+H]⁺=492.16. Found: 492.62.

Compound 9—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.25-7.20 (m,3H), 7.10-7.00 (m, 1H), 6.85 (d, J=6.00 Hz, 1H), 5.13 (d, J=11.10 Hz,1H), 4.30-4.10 (m, 2H), 2.72 (s, 3H), 2.02 (dd, J=15.30, 7.20 Hz, 1H),1.52 (dd, J=15.30, 3.90 Hz, 1H), 0.79 (s, 9H); ESI: Calculated forC₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.46.

Compound 9—Isomer B (TFA salt):

¹H NMR (300 MHz, CD₃OD): 7.68 (d, J=8.47 Hz, 1H), 7.32-7.16 (m, 3H),7.04 (d, J=7.63 Hz, 1H), 6.85 (d, J=6.01 Hz, 1H), 5.23 (d, J=11.21 Hz,1H), 4.46 (dd, J=8.23, 1.76 Hz, 1H), 4.13 (d, J=11.21 Hz, 1H), 2.71 (S,3H), 1.90 (dd, J=15.46, 8.23 Hz, 1H), 1.17 (dd, J=15.48, 1.75 Hz, 1H),0.89 (s, 9H)

¹³C (75 MHz, CD₃OD): 177.57, 168.31, 159.98 (d, J_(C-F)=244.73 Hz),140.90, 135.98, 134.31, 131.59, 130.56, 129.45, 128.47, 125.84 (d,J_(C-F)=7.48 Hz), 123.61 (d, J_(C-F)=19.45 Hz), 114.33 (d, J_(C-F)=25.29Hz), 113.57, 65.62, 64.15, 62.93, 57.04, 63.42, 31.01, 29.60, 27.02;ESI: Calculated for C₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.46.

Compound 9—Isomer C (TFA salt):

¹H NMR (300 MHz, CD₃OD): 7.66 (d, J=8.70 Hz, 1H), 7.32 (d, J=8.20 Hz,1H), 7.23 (t, J=7.89 Hz, 1H), 7.09 (t, J=1.70 Hz, 1H), 6.93 (d, J=7.66Hz, 1H), 6.89 (, J=6.13 Hz, 1H), 4.22 (t, J=5.73 Hz, 1H), 3.91 (d,J=12.36 Hz, 1H), 2.70 (s, 3H), 1.90-1.84 (m, 2H), 0.83 (s, 9H); ESI:Calculated for C₂₄H₂₇ ³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.58.

Compound 9—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.51 (d, J=8.79Hz, 1H), 7.24-7.12 (m, 2H), 7.08 (s, 1H), 7.25 (d, J=7.13 Hz, 1H), 6.85(d, J=6.13 Hz, 1H), 4.80 (d, J=11.25 Hz, 1H), 4.25-4.15 (m, 1H), 4.09(d, J=11.25 Hz, 1H), 2.76 (s, 3H), 1.44 (dd, J=15.26, 7.62 Hz, 1H), 1.08(dd, J=15.26, 3.19 Hz, 1H), 0.92 (s, 9H); ESI: Calculated for C₂₄H₂₇³⁵Cl₂FN₃O₂ [M+H]⁺=478.15. Found: 478.56.

Compound 10—Isomer A (TFA salt) (MI-2,9-TFA salt): ¹H NMR (300 MHz,CD₃OD): 7.32-7.22 (m, 3H), 7.18-7.10 (m, 1H), 7.06 (d, J=8.74 Hz, 1H),6.88 (d, J=6.08 Hz, 1H), 4.97 (d, J=10.79 Hz, 1H), 4.22 (d, J=10.79 Hz,1H), 4.26-4.18 (m, 1H), 3.50-3.20 (m, 5H), 2.03 (dd, J=15.35, 6.56 Hz,1H), 1.68-1.52 (m, 2H), 1.50-1.40 (m, 1H), 0.84 (s, 9H); ¹³C (75 MHz,CD₃OD): 179.64, 166.47, 140.40 (d, J_(P-C)=2.94 Hz), 136.54, 136.06,131.64, 130.16, 130.02, 128.14, 127.27 (d, J_(P-C)=40.14 Hz), 123.05 (d,J_(P-C)=19.44 Hz), 115.94 (d, J_(P-C)=25.54 Hz), 113.09, 70.91, 67.24,64.19, 64.10, 62.73, 57.52, 42.66, 38.14, 33.85, 30.99, 29.57; ESI:Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18. Found: 552.75; HPLCPurity=82%.

Compound 10—Isomer B, (TFA salt) (MI-21901—TFA salt): ¹H NMR (300 MHz,CD₃OD): 7.68 (d, J=8.47 Hz, 1H), 7.32-7.17 (m, 3H), 7.12-7.02 (m, 1H),6.85 (d, J=6.00 Hz, 1H), 5.23 (d, J=11.28 Hz, 1H), 4.46 (dd, J=8.41,1.91 Hz, 1H), 4.10 (d, J=11.28 Hz, 1H), 3.50-3.20 (m, 5H), 1.90 (dd,J=15.18, 8.62 Hz, 1H), 1.65-1.47 (m, 1H), 1.47-1.33 (m, 1H), 1.18 (d,J=15.18 Hz, 1H), 0.89 (s, 9H); 13C (75 MHz, CD₃OD): 177.56, 167.81,155.99 (d, J_(P-C)=243.27 Hz), 140.87, 135.98, 134.28, 131.63, 130.60,129.51, 128.60, 125.82 (d, J_(P-C)=7.39 Hz), 123.58 (d, J_(P-C)=19.68Hz), 114.30 (d, J_(P-C)=25.32 Hz), 113.55, 70.86, 67.23, 65.53, 64.16,63.07, 57.35, 43.43, 38.15, 33.78, 31.01, 29.57; ESI: Calculated forC₂₇H₃₃ ³⁵Cl₂FN₃O₄ [M+H]⁺=552.18. Found: 552.38; HPLC Purity=95%.

Compound 10—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.60-7.53 (m,1H), 7.30-7.10 (m, 2H), 7.08-7.02 (m, 1H), 7.00-6.92 (m, 1H), 6.84 (d,J=6.13 Hz, 1H), 4.96 (d, J=12.25 Hz, 1H), 4.39 (dd, J=7.22, 4.31 Hz,1H), 4.10 (d, J=12.27 Hz, 1H), 3.50-3.20 (m, 5H), 1.68 (dd, J=15.29,7.23 Hz, 1H), 1.62-1.48 (m, 1H), 1.48-1.32 (m, 1H), 1.17 (dd, J=15.29,4.16 Hz, 1H), 0.90 (s, 9H); ESI: Calculated for C₂₇H₃₃ ³⁵Cl₂FN₃O₄[M+H]⁺=552.18. Found: 552.40.

Compound 11—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.50-7.40 (m,2H), 7.28-7.18 (m, 1H), 6.91 (d, J=1.81 Hz, 1H), 6.78 (dd, J=8.13, 1.87Hz, 1H), 6.46 (d, J=8.14 Hz, 1H), 5.30 (d, J=8.52 Hz, 1H), 4.43 (d,J=8.52 Hz, 1H), 4.40-4.20 (m, 2H), 4.08 (dd, J=7.47, 2.43 Hz, 1H), 2.00(dd, J=15.32, 7.53 Hz, 1H), 1.22 (t, J=7.12 Hz, 3H), 1.19 (dd, J=15.32,2.55 Hz, 1H), 0.81 (s, 9H); ¹³C (75 MHz, CD₃Cl, note free amine):181.33, 171.91, 156.54 (d, J_(C-F)=248.02 Hz), 142.85, 134.15, 129.91,128.37 (d, J_(C-F)=14.61 Hz), 127.19 (d, J_(C-F)=3.19 Hz), 125.94,124.94, 124.63 (d, J_(C-F)=4.50 Hz), 122.10, 121.69 (d, J_(C-F)=18.50Hz), 110.72, 67.16, 65.70, 63.19, 61.74, 51.17, 43.45, 30.33, 29.97,14.32; ESI: Calculated for C₂₅H₂₈ ³⁵Cl₂FN₂O₃ [M+H]⁺=493.15. Found:493.44.

Compound 11—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.59 (d,J=7.41 Hz, 1H), 7.50-7.42 (m, 1H), 7.40-7.32 (m, 1H), 7.17-7.07 (m, 2H),6.78 (d, J=1.65 Hz, 1H), 5.61 (d, J=12.25 Hz, 1H), 4.56 (d, J=12.25 Hz,1H), 4.47 (dd, J=8.50, 1.50 Hz, 1H), 4.25 (dq, J=10.77, 7.12 Hz, 1H),4.13 (dq, J=10.77, 7.12 Hz, 1H), 1.93 (dd, J=15.39, 8.67 Hz, 1H), 1.34(dd, J=15.39, 1.57 Hz, 1H), 1.10 (t, J=7.12 Hz, 3H), 0.87 (s, 9H); ESI:Calculated for C₂₅H₂₈ ³⁵Cl₂FN₂O₃ [M+H]⁺=493.15. Found: 493.44.

Compound 12—Isomer A (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.56-7.46 (m,1H), 7.46-7.36 (m, 1H), 7.26-7.18 (m, 1H), 6.91 (d, J=1.61 Hz, 1H), 6.76(dd, J=8.10, 1.50 Hz, 1H), 6.47 (d, J=8.14 Hz, 1H), 5.29 (d, J=8.67 Hz,1H), 4.43 (d, J=8.67 Hz, 1H), 4.11 (d, J=7.47, 2.13 Hz, 1H), 2.02 (dd,J=15.36, 7.50 Hz, 1H), 1.16 (dd, J=15.36, 2.24 Hz, 1H), 0.81 (s, 9H);ESI: Calculated for C₂₃H₂₄ ³⁵Cl₂FN₂O₃ [M+H]⁺=465.11. Found: 465.42.

Compound 12—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.58-7.42 (m,2H), 7.36-7.26 (m, 1H), 7.14-7.02 (m, 1H), 6.75 (s, 1H), 5.43 (d,J=12.00 Hz, 1H), 4.58 (d, J=11.97 Hz, 1H), 4.35 (d, J=8.53 Hz, 1H), 1.87(dd, J=15.08, 9.31 Hz, 1H), 1.09 (d, J=15.31 Hz, 1H), 0.85 (s, 9H); ESI:Calculated for C₂₃H₂₄ ³⁵Cl₂FN₂O₃ [M+H]⁺=465.11. Found: 465.38.

Compound 12—Isomer D (TFA salt): ¹H NMR (300 MHz, CD₃OD): 7.49 (d,J=8.10 Hz, 1H), 7.36-7.26 (m, 1H), 7.13 (dd, J=8.10, 1.85 Hz, 1H),7.08-7.00 (m, 1H), 6.96-6.86 (m, 1H), 6.78 (d, J=1.80 Hz, 1H), 5.32 (d,J=12.69 Hz, 1H), 4.56 (d, J=12.69 Hz, 1H), 4.42 (dd, J=7.27, 3.88 Hz,1H), 1.69 (dd, J=15.44, 7.64 Hz, 1H), 1.13 (dd, J=15.44, 3.76 Hz, 1H),0.90 (s, 9H); ESI: Calculated for C₂₃H₂₄ ³⁵Cl₂FN₂O₃ [M+H]⁺=465.11.Found: 465.54.

Example 17 Synthesis of CB061—Isomer B (TFA Salt)

CB061 was prepared according to Scheme 12 using methodology described inEXAMPLE 16 to give an A:B:(C+D) isomer ratio after oxidation of15:67:18, an A:B:(C+D) isomer ratio after isomerization of 2:74:24, andsubstantially pure CB061—Isomer B (as the TFA salt) after chromatography(FIG. 30).

Analytical data for CB061—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD):7.66 (d, J=8.45 Hz, 1H), 7.30-7.16 (m, 3H), 7.10-7.03 (m, 1H), 6.85 (d,J=5.98 Hz, 1H), 5.22 (d, J=11.37 Hz, 1H), 4.46 (dd, J=8.31, 1.68 Hz,1H), 4.09 (d, J=11.37 Hz, 1H), 3.70-3.50 (m, 1H), 3.50-3.39 (m, 1H),2.00-1.84 (m, 3H), 1.82-1.70 (m, 1H), 1.58-1.46 (m, 1H), 1.40-1.12 (m,4H), 1.08-0.90 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, CD₃OD): 177.42,166.80, 155.82 (d, J_(C-F)=243.61 Hz), 140.67 (d, J_(C-F)=2.77 Hz),135.73, 134.18, 131.36, 130.33, 129.44, 128.37, 125.77 (d, J_(C-F)=7.38Hz), 123.38 (d, J_(C-F)=19.54 Hz), 114.06 (d, J_(C-F)=25.18 Hz), 113.39,69.97, 65.12, 64.06, 62.94, 57.41, 49.88, 43.37, 34.38, 34.29, 30.98,30.82, 29.39; ESI: Calculated for C₂₉H₃₅Cl₂FN₃O₃ [M+H]⁺=562.20. Found:562.36. The absolute stereochemistry of CB061—Isomer B was determined bysingle crystal x-ray crystallography.

Example 18 Synthesis of CB087—Isomer B (TFA Salt)

CB087 was prepared according to Scheme 13 using methodology described inEXAMPLE 16 to give an (A+C+D):B isomer ratio after oxidation of 65:35,an (A+C+D):B isomer ratio after isomerization of 18:82 (inacetonitrile-water in the presence of TFA), and substantially pureCB087—Isomer B (as the TFA salt) after chromatography. In this study,intermediate A was isolated as an 85:15 mixture of isomers based on ¹HNMR analysis. This mixture was used in the oxidation step. The majorisomer was characterized by ¹H NMR and ESI. Also, little isomerizationwas observed in MeOH or in MeOH-water at a pH of 8 after 3 days at roomtemperature.

Analytical data for compound A major isomer: ¹H NMR (300 MHz, CD₃OD):7.40 (s, 6H), 7.10 (s, 9H), 7.20 (t, J=7.12 Hz, 1H), 6.74 (t, J=7.86 Hz,1H), 6.48 (s, 1H), 6.44 (d, J=8.20 Hz, 1H), 6.08 (t, J=7.02 Hz, 1H),5.51 (d, J=8.22 Hz, 1H), 5.35 (d, J=4.67 Hz, 1H), 4.55 (d, J=4.45 Hz,1H), 4.18 (s, 1H), 4.15-3.90 (m, 3H), 3.70-3.55 (m, 1H), 2.30-2.00 (m,4H), 1.60-1.30 (m, 4H); ¹³C (75 MHz, CD₃OD): 179.68, 175.39, 155.86 (d,J_(C)-P=247.63 Hz), 142.81, 134.49, 134.21, 132.34, 132.09, 129.46,129.28 (d, J_(C-P)=12.91 Hz), 128.45, 128.09, 127.78, 127.16, 126.90,126.67, 125.20, 124.90, 124.08 (d, J_(C-P)=3.27 Hz), 121.32 (d,J_(C-P)=8.18 Hz), 121.17, 110.01, 76.10, 75.35, 69.39, 67.82, 66.89,61.60, 49.60, 48.14, 33.60, 33.48, 30.12, 29.82; ESI: Calculated forC₄₄H₄₁Cl₂FN₃O₄ [M+H]⁺=764.25. Found: 764.26.

Analytical data for CB087—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD):8.34 (d, J=7.70 Hz, 1H), 7.70 (d, J=8.11 Hz, 1H), 7.70 (t, J=7.02 Hz,1H), 7.42 (t, J=7.61 Hz, 1H), 7.40-7.30 (m, 5H), 7.22 (d, J=7.90 Hz,1H), 7.16 (dd, J=8.17, 1.85 Hz, 1H), 6.61 (d, J=1.74 Hz, 1H), 5.58 (s,1H), 5.34 (d, J=10.97 Hz, 1H), 4.83 (d, J=11.04 Hz, 1H), 3.78-3.60 (m,1H), 3.50-3.40 (m, 1H), 2.00-1.86 (m, 2H), 1.86-1.78 (m, 1H), 1.70-1.60(m, 1H), 1.42-1.20 (m, 3H), 1.02 (qd, J=12.68, 3.30 Hz, 1H); ¹³C (75MHz, CD₃OD): 177.29, 167.33, 154.20 (d, J_(C-F)=284.04 Hz), 144.70,136.97, 132.50, 131.26, 130.24, 130.03, 128.83, 128.70, 126.61, 126.54,123.98, 122.91, 122.35 (d, J_(C-F)=18.95 Hz), 121.70 (d, J_(C-F)=12.66Hz), 111.78, 70.44, 69.95, 64.89, 62.47, 49.98, 49.87, 34.33, 34.28,30.91, 30.80; ESI: Calculated for C₃₀H₂₉Cl₂FN₃O₃ [M+H]⁺=568.16. Found:568.54.

Example 19 Synthesis of CB083—Isomer B (TFA Salt)

CB083 was prepared according to Scheme 14 using methodology described inEXAMPLE 16 to give an (A+C+D):B isomer ratio after oxidation of 41:59,an (A+C+D):B isomer ratio after isomerization of 10:90 (inacetonitrile-water in the presence of TFA), and substantially pureCB083—Isomer B (as the TFA salt) after chromatography. In this study,intermediate A was isolated as an 84:16 mixture of isomers, and themajor isomer was used in the oxidation step. Also, little isomerizationwas observed in MeOH or in MeOH-water at a pH of 8 after 3 days at roomtemperature.

Analytical data for compound A major isomer: ¹H NMR (300 MHz, CD₃OD):8.42 (d, J=7.49 Hz, NH, 1H), 7.40-7.25 (m, 5H), 7.25-7.10 (m, 9H),7.20-6.98 (m, 3H), 6.94 (dd, J=8.06, 1.88 Hz, 1H), 6.82 (d, J=8.07 Hz,1H), 6.63-6.55 (m, 2H), 6.53 (d, J=1.84 Hz, 1H), 5.18 (d, J=5.70 Hz,1H), 4.32 (s, 1H), 3.97 (d, J=5.70 Hz, 1H), 3.70 (d, J=7.57 Hz, 1H),3.70-3.55 (m, 2H), 3.25-3.10 (m, 1H), 2.64 (dd, J=14.03, 6.91 Hz, 1H),2.45 (dd, J=13.97, 9.24 Hz, 1H), 2.15-1.90 (m, 4H), 1.55-1.30 (m, 4H);¹³C (75 MHz, CD₃OD): 181.03, 176.38, 144.79, 144.55, 139.09, 138.54,135.17, 134.94, 133.16, 130.13, 130.07, 129.77, 129.26, 129.11, 129.03,128.95, 128.80, 128.18, 127.66, 127.35, 122.68, 111.27, 79.40, 76.05,72.19, 70.6, 70.56, 62.05, 53.09, 49.63, 37.84, 34.88, 34.77, 31.34,31.07; ESI: Calculated for C₄₅H₄₅ClN₃O₄ [M+H]⁺=726.31. Found: 726.44.

Analytical data for CB083—Isomer B (TFA salt): ¹H NMR (300 MHz,DMSO-d₆): 10.28 (s, NH, 1H), 8.51 (d, J=6.67 Hz, NH, 1H), 7.59 (d,J=8.03 Hz, 1H), 7.25-7.00 (m, 9H), 6.60-6.50 (m, 3H), 4.82 (s, 1H), 4.42(d, J=9.65 Hz, 1H), 3.64-3.50 (m, 1H), 3.46-3.32 (m, 1H), 3.30-3.16 (m,1H), 2.85 (dd, J=13.83, 4.77 Hz, 1H), 2.28 (dd, J=13.83, 10.20 Hz, 1H),1.90-1.66 (m, 4H), 1.38-1.10 (m, 4H); ¹³C (75 MHz, DMSO-d6): 178.36,166.47, 143.95, 136.55, 133.32, 131.90, 128.69, 128.46, 128.09, 127.88,127.18, 126.83, 126.52, 123.21, 120.72, 109.97, 70.38, 67.78, 63.18,62.17, 52.83, 47.76, 34.54, 33.57, 29.82, 29.78; ESI: Calculated forC₃₁H₃₃ClN₃O₃ [M+H]⁺=530.22. Found: 530.40.

Example 20 Synthesis of Synthesis of CB084—Isomer B (TFA Salt)

CB084 was prepared according to Scheme 15 using methodology described inEXAMPLE 16 to give an A:B:(C+D) isomer ratio after oxidation of 97:1:2,an A:B:(C+D) isomer ratio after isomerization of 46:52:2 inacetonitrile-water in the presence of TFA after 3 days, an A:B:(C+D)isomer ratio after isomerization of 9:49:42 in acetonitrile-water at pH8 after 3 days, and substantially pure CB084—Isomer B (as the TFA salt)after chromatography.

Analytical data for CB084—Isomer A (free amine): ¹H NMR (300 MHz,DMSO-d6): 10.02 (s, NH, 1H), 7.86 (d, J=7.60 Hz, NH, 1H), 7.31 (d,J=7.91 Hz, 1H), 7.05 (d, J=7.91 Hz, 1H), 6.97 (s, 3H), 6.54 (s, 1H),6.46 (d, J=3.65 Hz, 2H), 4.50 (d, J=4.08 Hz, NH, 1H), 3.60-3.40 (m, 2H),3.40-3.30 (m, 1H), 3.25-3.10 (m, 1H), 2.80-2.70 (m, 2H), 2.60-2.40 (m,1H), 1.90-1.60 (m, 4H), 1.34 (dd, J=13.86, 10.33 Hz, 1H), 1.30-1.05 (m,4H), 0.90-0.70 (m, 1H), 0.71 (s, 9H); ¹³C (75 MHz, DMSO-d6): 180.50,169.86, 143.83, 138.61, 131.88, 129.53, 127.91, 127.64, 125.83, 125.55,120.61, 108.90, 68.15, 68.08, 68.03, 61.27, 55.94, 47.20, 43.56, 34.36,33.83, 33.77, 30.30, 30.02, 29.80, 29.67; ESI: Calculated for C₃₀H₃₉³⁵ClN₃O₃ [M+H]⁺=524.27. Found: 524.55.

Analytical data for CB084—Isomer B (TFA salt): ¹H NMR (300 MHz, CD₃OD):8.54 (d, J=7.42 Hz, NH, 1H), 7.48 (d, J=8.06 Hz, 1H), 7.23 (d, J=8.06Hz, 1H), 7.20-7.08 (m, 3H), 6.90 (s, 1H), 6.67 (d, J=6.94 Hz, 1H), 4.49(d, J=11.08 Hz, 1H), 4.17 (dd, J=7.91, 1.68 Hz, 1H), 3.75-3.60 (m, 1H),3.60-3.45 (m, 1H), 3.40-3.30 (m, 1H), 2.79 (dd, J=14.10, 6.05 Hz, 1H),2.36 (dd, J=14.10, 10.21 Hz, 1H), 2.10-1.80 (m, 4H), 1.49 (dd, J=15.58,8.30 Hz, 1H), 1.45-1.20 (m, 4H), 1.00-0.80 (m, 1H), 0.87 (s, 9H); ¹³C(75 MHz, DMSO-d6): 175.40, 144.42, 136.34, 133.82, 128.42, 127.93,127.02, 126.60, 123.34, 121.24, 110.53, 67.72, 63.74, 63.35, 61.30,51.75, 47.89, 42.56, 33.47, 33.44, 32.92, 29.78, 29.66, 29.52, 28.85;ESI: Calculated for C₃₀H₃₉ ³⁵ClN₃O₃ [M+H]⁺=524.27. Found: 524.44.

Example 21 Synthesis of C144

C144 was prepared using methodology described above. The absolutestereochemistry of C144 determined by single crystal x-raycrystallography.

Analytical data for C144: ¹H NMR (300 MHz, CD₃Cl): 7.70-7.40 (m, 5H),7.50-7.00 (m, 9H), 7.00-6.80 (m, 5H), 6.80-6.60 (m, 2H), 6.43 (d, J=7.68Hz, 1H), 6.11 (t, J=5.79 Hz, 1H), 5.99 (d, J=7.56 Hz, 1H), 5.27 (d,J=6.91 Hz, 1H), 4.48 (dd, J=14.79, 6.31 Hz, 1H), 4.34 (d, J=6.91 Hz,1H), 4.21 (dd, J=14.79, 5.41 Hz, 1H), 4.13 (d, J=11.24 Hz, 1H), 3.80 (d,J=11.24 Hz, 1H), 3.51 (d, J=9.46 Hz, 1H), 2.83 (dd, J=15.40, 9.46 Hz,1H), 1.32 (d, J=15.40 Hz, 1H), 0.76 (s, 9H); ¹³C (75 MHz, CD₃Cl):178.42, 173.63, 142.12, 139.27, 137.97, 134.54, 133.23, 132.49, 131.78,129.21, 128.88, 128.74, 128.48, 128.21, 127.92, 127.86, 127.77, 127.52,127.29, 124.63, 122.27, 109.35, 75.06, 72.14, 70.40, 66.08, 60.65,60.51, 43.62, 43.56, 30.11, 29.91; ESI: Calculated for C₄₄H₄₆N₃O₃[M+H]⁺=664.35. Found: 664.36.

Example 22 Cellular Activity of MI-77301

The activity of MI-773001 in a variety of tumor cell lines is presentedin Table 6. The cytotoxic concentration is the first concentration wherecell death corresponding to the cytotoxic index is observed.Cytotoxicity was quantified by blue trypan exclusion at 96 h except forCCF-STTG1 at 192 hours (medium 20-50% cell death and high >50% celldeath). The anti-proliferative IC₅₀ was determined by ATP assay.

TABLE 6 tumor Cytotox* IC₅₀ cell line origin p53 BM Cytotox (μM) (nM)H1299 lung —/— — — — >10,000 U2OS bone WT/WT — — — >10,000 SJSA1 boneWT/WT MDM2 high 1 145 amplified HCT116 colon WT/WT — — — 229 RKO colonWT/WT — — — 480 22RV1 prostate WT/MUT — medium 3 399 LnCap prostateWT/WT — high 3 50 JAR placenta WT/WT MDM2 high 3 169 amplified CCF- CNSWT/WT MDM2 medium 1 162 STTG1 amplified Capan2 pancreas —/— — —— >10,000 MCF7 breast WT/WT — — — 201 Y79 retino- WT/WT Rb medium 1 430blastome mutated Weri- retino- WT/WT Rb high 0.3 not RB1 blastomemutated determined SNU-1 gastric WT/WT KRAS high 10 77 mut MKN45 gastricWT/WT Met — — >10,000 amplified Hs746T gastric Met — — 10,000 amplifiedSNU-5 gastric Met — — >10,000 amplified MOLM13 AML WT/WT FLT3-ITD high0.1 14.7 MV4; 11 AML WT/WT FLT3-ITD medium 0.3 45.4 RS4; 11 ALL WT/WT —high 0.1 28 LY3 ABC- WT/WT — medium 0.3 280 DLBCL Ly10 ABC- WT/WT —medium/ 0.3 90 DLBCL high Ly7 GCB- WT/MUT — — — >10,000 DLBCL DoHH2 GCB-WT/WT BCL2 high 0.3 39.3 DLBCL overexpression Rec1 MCL WT/WT — high 10738 SR lymphoma WT/WT — medium 0.3 14.9 H929 MM WT/WT — high 1 114 Ku812CML-BC WT/WT — medium 3 >10,000 EHEB CLL WT/WT — medium 0.3 70

Example 23 Cell Growth Inhibition and Cytotoxic Effects on 22Rv1 CellLines

MI-519-6401 and MI-77301 were evaluated for their cell growth inhibitionand cytotoxic effects on a prostate cancer cell line 22Rv1 (from TheDSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,reference DSMZ ACC438). For growth inhibition assays, cells wereincubated in the presence of MI-519-6401 or MI-77301 for 96 h in 96-wellformat. Cell seeding conditions were adapted to get significant cellgrowth in this assay format. Growth inhibition assays were performedusing the Celltiter-Glo Luminescent kit (Promega). The IC₅₀ values(concentration where the growth inhibition percentage is equal to halfof the maximum inhibitory effect of the tested compound) were calculatedand ranged between 100 nM and 500 nM in this prostate cancer cell linefor both compounds.

For cytotoxicity assays, cells were incubated with MI-519-6401 orMI-77301 for 96 h in 6-well format. Cell seeding conditions were adaptedto get significant cell growth in this assay format. Cytotoxic effectswere measured using trypan blue staining. Both the floating and adherentcells were stained with trypan blue. Quantification was performed byVi-CELL® Cell Viability Analyzer (Beckmann-Coulter) which determinesboth cell concentration and percentage of viable cells. For bothcompounds at concentrations which were close to IC₉₀ concentrations(concentration where the growth inhibition percentage is equal to 90percent of the maximum inhibitory effect of the tested compound), thepercentages of viable cells were significantly decreased compared tountreated cells and were at best between 50 and 70% in the 22Rv1 cellline.

Example 24 Apoptosis Assay

Apoptosis was determined using Annexin-V-FLUOS/propidium iodide stainingkit (Roche Applied Science) by modifications of the manufacturer'sinstructions. Early stage apoptotic cells display translocation ofphosphatidylserine from inner to outer surface of plasma membrane whichcan be detected by Annexin V fluorescein staining. Propidium iodide (PI)staining determines the late stage apoptotic cells or necrotic cells. Atotal of 0.25×10⁶ cells were plated overnight in a 6-well tissueculture. Next day, adherent cells were treated in the presence orabsence of MI-77301 and incubated at 37° C. for 2.5 days. Cells wereharvested using 0.05% trypsin-EDTA (Invitrogen) by pooling the floatingand the adherent cell populations, and were washed with PBS. Next, cellswere stained with Annexin-V-FLUOS and PI in the incubation buffer atroom temperature for 10 minutes in the dark. Modifications were made byusing 0.5 μl each of Annexin-V-FLUOS and PI (instead of 2 μl each,mentioned in the instruction manual) for staining. These modificationswere found to decrease the background noise, especially of fluorescein,in the assay. Cells were acquired and analyzed in a flow cytometer.Annexin V+/PI− cells were scored as early apoptotic cells and AnnexinV+/PI+ cells as late apoptotic cells. The results of this assay arepresented in FIG. 27.

Example 25 In Vivo Efficacy in the SK-Mel-103 Xenograft Model in MiceDrug Preparation

MI-77301 was dissolved in 10% PEG400 (polyethylene glycol, mol wt 400,Sigma #P3265) 3% Cremophor EL (Sigma #C5135) and 87% 1×PBS (GIBCO™,Invitrogen Corp.) at the desired concentration (prepared fresh each dayand dosed orally within 1 hour). The pH of the drug solutions werechecked before use and required to be between pH 3.0 and 9.0 for PO(oral gavage) and between pH 4.5 and 9.0 for IV (intravenous)administration. The pH of a solution was adjusted with 0.5N NaOH whennecessary.

Cell Culture

Human melanoma cells SK-MEL-103 were maintained at 37° C., 95% air, 5%carbon dioxide in HyQ® RPMI-1640 medium (with 2.05 mM L-glutamine, 0.1μM sterile filtered, Hyclone®, QB Perbio) supplemented with 10% FBS andpenicillin/streptomycin and passaged twice weekly.

Xenograft Tumor Cell Injection

Tumor cells for xenografts were harvested with Trypsin (0.05%)-EDTA(0.53 mM) (GIBCO™, Invitrogen Corp.), growth medium added and cellsplaced on ice. A cell sample was mixed 1:1 with Trypan Blue (GIBCO™,Invitrogen Corp.) and counted on a hemocytometer to determine the numberof live/dead cells. Cells were washed once with 1×PBS (GIBCO™,Invitrogen Corp.) and resuspended in PBS. Cells in 0.1 ml were injectedsubcutaneously (s.c.) into the flank region of each mouse using a 27gauge needle. For Matrigel injections, after washing in PBS, cells wereresuspended in an ice cold mixture of 1:1 PBS and Matrigel (BDBiosciences, Invitrogen Corp.) for a final Matrigel proteinconcentration of 5 mg/ml. SK-MEL-103 tumors were inoculated into SCIDmice (UM breeding strain: 236 C.B-17 SCID, Charles River) at 5×10⁶ cellsin 0.1 ml with Matrigel. Treatment was started on day 4-5 after tumorinjection.

Xenograft Tumor Growth and Weight Monitoring

The size of tumors growing in the mice was measured in two dimensionsusing calipers. Tumor volume (mm³)=(A×B²)/2 where A and B are the tumorlength and width (in mm), respectively. During treatment, tumor volumeand body weight were measured three times a week. After the treatmentwas stopped, tumor volume and body weight was measured at least once aweek and mice were kept for additional 60 days for further observationof tumor growth and toxicity.

Assessment of Toxicity and End Point

Tumors were not allowed to exceed 10% of the animal's total body weight.If an animal had two or more tumors the total weight of all tumors werenot allowed to exceed 10% of the animal's total body weight. At the endof the experimental period or when tumor size approached 10% of thetotal body weight, the animal was euthanized. Animals that showedprofound morbidity or a weight loss of over 20% of body weight wereeuthanized.

The efficacy of MI-77301 at 100 mg/kg PO in this assay is presented inFIG. 29.

Having now fully described the methods, compounds, and compositions ofmatter provided herein, it will be understood by those of skill in theart that the same can be performed within a wide and equivalent range ofconditions, formulations, and other parameters without affecting thescope of the methods, compounds, and compositions provided herein or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A compound having Formula XII:

wherein: R^(1a), R^(1b), R^(1c), and R^(1d) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro; R²is selected from the group consisting of aralkyl and:

wherein: R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are eachindependently selected from the group consisting of hydrogen, fluoro,and chloro; R³ is selected from the group consisting of optionallysubstituted C₁-C₈ alkyl and optionally substituted aryl; R⁴ is selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl; R⁵ is selected from the group consisting of:

wherein: R¹⁴ is selected from the group consisting of hydrogen andoptionally substituted C₁-C₄ alkyl; X is selected from the groupconsisting of O, S, and NR′; Y is selected from the group consisting ofO, S, and NR″; R′ is selected from the group consisting of hydrogen andoptionally substituted C₁-C₄ alkyl; and R″ is selected from the groupconsisting of hydrogen, optionally substituted C₁-C₄ alkyl, and —COCH₃,wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1, wherein: R² is:

R³ is optionally substituted C₁-C₈alkyl; R⁵ is selected from the groupconsisting of:

and R″ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₄ alkyl, or a pharmaceutically acceptable salt thereof.3. The compound of claim 2, wherein R⁴ is hydrogen, or apharmaceutically acceptable salt thereof.
 4. The compound of claim 2,wherein X is NH, or a pharmaceutically acceptable salt thereof.
 5. Thecompound of claim 2, wherein Y is NH, or a pharmaceutically acceptablesalt thereof.
 6. The compound of claim 2, wherein R³ is —CH₂C(CH₃)₃, ora pharmaceutically acceptable salt thereof.
 7. The compound of claim 2,wherein R⁵ is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim2, wherein: R^(1a) is hydrogen; R^(1b), R^(1c), and R^(1d) are eachindependently selected from the group consisting of hydrogen, fluoro,and chloro; R³ is C₄-C₈ alkyl; R⁴ is hydrogen; R⁵ is selected from thegroup consisting of:

and X and Y are NH; or a pharmaceutically acceptable salt thereof. 9.The compound of claim 8, wherein R⁵ is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 10. A compound havingFormula XXXV:

wherein: R^(1b) and R^(1c) are independently selected from the groupconsisting of hydrogen, fluoro, and chloro; R³ is C₄-C₈ alkyl; andR^(25a), R^(25b), and R^(25c) are each independently selected from thegroup consisting of hydrogen, fluoro, and chloro, wherein the compoundis substantially free of one or more other stereoisomers, or apharmaceutically acceptable salt thereof.
 11. The compound of claim 2selected from the group consisting of:

wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 12. Thecompound of claim 1 selected from the group consisting of:

wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 13. Acompound selected from the group consisting of:

wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 14. Acompound having the structure:

wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 15. Acompound having the structure:

wherein the compound is substantially free of one or more otherstereoisomers, or a pharmaceutically acceptable salt thereof.
 16. Thecompound of claim 1, wherein the compound is a substantially purestereoisomer, or a pharmaceutically acceptable salt thereof. 17.(canceled)
 18. A pharmaceutical composition comprising the compound ofclaim 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 19. (canceled)
 20. A method oftreating a patient comprising administering to the patient atherapeutically effective amount of the compound of claim 1, or apharmaceutically acceptable salt thereof, wherein the patient has ahyperproliferative disease. 21-24. (canceled)
 25. The method of claim20, wherein the hyperproliferative disease is cancer. 26-28. (canceled)29. The method of claim 25, wherein the cancer is selected from thegroup consisting of melanoma, lung cancer, sarcoma, colon cancer,prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomachcarcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, andleukemia. 30-32. (canceled)
 33. The method of claim 29, wherein thecancer is selected from the group consisting of liposarcoma andmelanoma. 34-36. (canceled)
 37. The method of claim 20, furthercomprising administering to the patient one or more anticancer agents.38-39. (canceled)
 40. The method of claim 20, wherein the methodcomprises pulsatile dose administration of the compound, orpharmaceutically acceptable salt thereof, to the patient. 41-45.(canceled)
 46. A kit comprising the compound of claim 1, or apharmaceutically acceptable salt thereof, and instructions foradministering the compound, or a pharmaceutically acceptable saltthereof, to a patient having cancer. 47-51. (canceled)
 52. A method ofpreparing a compound having Formula XXXVII:

the method comprising allowing a compound having Formula XXXVI:

to isomerize to a compound having Formula XXXVII, wherein: R³² isselected from the group consisting of —OR³³ and —NR^(34a)R^(34b); R³³ isselected from the group consisting of hydrogen, alkyl, and aralkyl;R^(34a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, aralkyl,optionally substituted aryl, and optionally substituted heteroaryl;R^(34b) is selected from the group consisting of hydrogen and alkyl;R^(1a), R^(1b), R^(1c), and R^(1d) are each independently selected fromthe group consisting of hydrogen, fluoro, and chloro; R² is selectedfrom the group consisting of aralkyl and:

R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are each independentlyselected from the group consisting of hydrogen, fluoro, and chloro; andR³ is selected from the group consisting of optionally substituted C₁-C₈alkyl and optionally substituted aryl. 53-67. (canceled)